Use of semaphorin-4d inhibitory molecules in combination with an immune modulating therapy to inhibit tumor growth and metastases

ABSTRACT

Provided herein are methods for inhibiting, delaying, or reducing tumor growth and metastases of Plexin-B1-expressing cancer cells in a subject, comprising administering to the subject an effective amount of an isolated binding molecule which specifically binds to semaphorin-4D (SEMA4D) in combination with an effective amount of at least one other immune modulating therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation Application of currently pending U.S. applicationSer. No. 14/892,099 (allowed), filed Nov. 18, 2015, which is a U.S.National Stage of International Patent Application PCT/US2014/043466,filed on Jun. 20, 2014, which claims the benefit of U.S. ProvisionalAppl. No. 61/907,845, filed on Nov. 22, 2013, U.S. Provisional Appl. No.61/884,771, filed on Sep. 30, 2013, U.S. Provisional Appl. No.61/874,241, filed on Sep. 5, 2013, and U.S. Provisional Appl. No.61/839,170, filed on Jun. 25, 2013, and is related to U.S. patentapplication Ser. No. 14/310,848 filed on Jun. 20, 2014, now U.S. Pat.No. 9,243,068, the contents of each of which are hereby incorporated byreference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in the textfile (Name: 58008_168142_SEQ_LST.txt; Size: 37,189 bytes; and Date ofCreation: Jul. 28, 2017) filed with the application is incorporatedherein by reference in its entirety.

BACKGROUND

Semaphorin 4D (SEMA4D), also known as CD100, is a transmembrane protein(e.g., SEQ ID NO: 1 (human); SEQ ID NO: 2 (murine)) that belongs to thesemaphorin gene family. SEMA4D is expressed on the cell surface as ahomodimer, but upon cell activation SEMA4D can be released from the cellsurface via proteolytic cleavage to generate sSEMA4D, a soluble form ofthe protein, which is also biologically active. See Suzuki et al.,Nature Rev. Immunol. 3:159-167 (2003); Kikutani et al., Nature Immunol.9:17-23 (2008).

SEMA4D is expressed at high levels in lymphoid organs, including thespleen, thymus, and lymph nodes, and in non-lymphoid organs, such as thebrain, heart, and kidney. In lymphoid organs, SEMA4D is abundantlyexpressed on resting T cells but only weakly expressed on resting Bcells and antigen-presenting cells (APCs), such as dendritic cells(DCs). Its expression, however, is upregulated in these cells followingactivation by various immunological stimuli. The release of solubleSEMA4D from immune cells is also increased by cell activation. SEMA4Dhas been implicated in the development of certain cancers (Ch'ng et al.,Cancer 110:164-72 (2007); Campos et al., Oncology Letters, 5:1527-35(2013); Kato et al., Cancer Sci. 102:2029-37 (2011)) and several reportssuggest that one mechanism of this influence is the role of SEMA4D inpromoting tumor angiogenesis (Conrotto et al., Blood 105:4321-4329(2005). Basile et al., J Biol. Chem. 282: 34888-34895 (2007); Sierraet.al. J. Exp. Med. 205:1673 (2008); Zhou et al., Angiogenesis15:391-407 (2012)). Tumor growth and metastasis involve a complexprocess of cross talk amongst the tumor cells, stroma and immuneinfiltrate, as well as the endothelial cells and vasculature. SEMA4D isover-expressed in a wide array of tumor types and is also produced byinflammatory cells recruited to the tumor microenvironment, the questionof what role SEMA4D can play in migration, survival, differentiation andorganization of the different cell types that constitute the tumorstroma remains to be addressed.

SUMMARY

This application addresses the need for safe and effective cancertreatments that serve either as a single agent that inhibits, reduces,suppresses, prevents, slows or delays the progression of, shrinks, ordirectly attacks tumor cells or that can act in combination with otherimmune modulating therapies to enhance their therapeutic benefit. Inparticular, SEMA4D was shown to play a role in the infiltration,maturation and organization of immune cells and macrophage that eitherpromote or inhibit tumor growth, which can contribute to development ofeffective methods for reducing tumor growth and metastases in a subjectwith cancer.

Certain aspects of the application are directed to a method forinhibiting, delaying, or reducing tumor growth or metastases or bothtumor growth and metastases in a subject with cancer comprisingadministering to the subject an effective amount of an isolated bindingmolecule which specifically binds to semaphorin-4D (SEMA4D) and aneffective amount of at least one other immune modulating therapy.

In some embodiments, the binding molecule inhibits SEMA4D interactionwith its receptor (e.g., Plexin-B1). In some embodiments, the bindingmolecule inhibits SEMA4D-mediated Plexin-B1 signal transduction. In someembodiments, the inhibition, delay, or reduction of metastases occursindependently of primary tumor growth inhibition, delay, or reduction.In some embodiments, the cancer is selected from the group consisting ofcarcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, gastric cancer,pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer,ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, esophageal cancer, salivary gland carcinoma, kidney cancer,liver cancer, prostate cancer, vulval cancer, thyroid cancer, head andneck cancer, and a combination thereof In some embodiments, the subjecthas elevated levels of either B cells, T cells or both B cells and Tcells when compared to other cancer subjects.

In some embodiments, the isolated binding molecule specifically binds tothe same SEMA4D epitope as a reference monoclonal antibody selected fromthe group consisting of VX15/2503 and 67. In some embodiments, theisolated binding molecule comprises an antibody or antigen-bindingfragment thereof In some embodiments, the antibody or antigen-bindingfragment thereof comprises the six complementarity determining regions(CDRs) of monoclonal antibody VX15/2503 or 67.

In some embodiments, the immune modulating therapy is selected from thegroup consisting of a cancer vaccine, an immunostimulatory agent,adoptive T cell or antibody therapy, immune checkpoint blockade and acombination thereof. In some embodiments, the immune modulating agent isselected from the group consisting of interleukins, cytokines,chemokines, antagonists of immune checkpoint blockades and a combinationthereof. In some embodiments, the immune modulating therapy can be acancer therapy. In some embodiments, the cancer therapy is selected fromthe group consisting of surgery or surgical procedures, radiationtherapy, chemotherapy or a combination thereof. In some embodiments, theisolated binding molecule and the immune modulating agent or immunemodulating therapy are administered separately or concurrently.

In some embodiments, methods for inhibiting, delaying, or reducing tumorgrowth in a subject with cancer are provided that comprise administeringto the subject an effective amount of an isolated binding molecule whichspecifically binds to semaphorin-4D (SEMA4D) and an effective amount ofat least one other immune modulating therapy. In some embodiments, thebinding molecule inhibits SEMA4D interaction with its receptor. In someembodiments, the receptor is Plexin-B1. In some embodiments, the bindingmolecule inhibits SEMA4D-mediated Plexin-B1 signal transduction. In someembodiments, the cancer is selected from the group consisting ofcarcinoma, lymphoma, blastoma, sarcoma, leukemia, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, gastric cancer,pancreatic cancer, neuroendocrine cancer, glioblastoma, cervical cancer,ovarian cancer, liver cancer, bladder cancer, brain cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, esophageal cancer, salivary gland carcinoma, kidney cancer,liver cancer, prostate cancer, vulval cancer, thyroid cancer, head andneck cancer, and a combination thereof In some embodiments, the isolatedbinding molecule specifically binds to the same SEMA4D epitope as areference monoclonal antibody VX15/2503 or 67. In some embodiments, theisolated binding molecule competitively inhibits a reference monoclonalantibody VX15/2503 or 67 from specifically binding to SEMA4D. In someembodiments, the isolated binding molecule comprises an antibody orantigen-binding fragment thereof. In some embodiments, the antibody orantigen-binding fragment thereof comprises a variable heavy chain (VH)comprising VHCDRs 1-3 comprising SEQ ID NOs 6, 7, and 8, respectively,and a variable light chain (VL) comprising VLCDRs 1-3 comprising SEQ IDNOs 14, 15, and 16, respectively. In some embodiments, the VH and VLcomprise, respectively, SEQ ID NO: 9 and SEQ ID NO: 17 or SEQ ID NO: 10and SEQ ID NO: 18. In some embodiments, the immune modulating therapy isselected from the group consisting of administration of a cancervaccine, administration of an immunostimulatory agent, adoptive T cellor antibody therapy, administration of an immune checkpoint blockadeinhibitor, administration of a regulatory T cell (Treg) modulator, and acombination thereof In some embodiments, the immune modulating therapycomprises an immune checkpoint blockade inhibitor. In some embodiments,wherein the immune checkpoint blockade inhibitor is an anti-CTLA4antibody, an anti-PD-1 antibody, or a combination thereof In someembodiments, the immune modulating therapy comprises administration of acancer vaccine. In some embodiments, the Treg modulator iscyclophosphamide. In some embodiments, the isolated binding molecule andthe immune modulating therapy are administered separately orconcurrently. In some embodiments, administration of the combination ofthe isolated binding molecule and the immune modulating therapy resultsin enhanced therapeutic efficacy relative to administration of theisolated binding molecule or the immune modulating therapy alone. Insome embodiments, the subject has an elevated level of B cells, T cellsor both B cells and T cells when compared to other cancer subjects. Insome embodiments, the level of B cells and/or T cells per microliter ofblood in the subject is about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 timesthe mean number of B cells and/or T cells in circulation in other cancerpatients. In some embodiments, the level of B cells and/or T cells permicroliter of blood in the subject ranges from about 147 to about 588and from about 1173 to about 3910, respectively, e.g., when compared toother cancer patients. In some embodiments, the subject has B celland/or T cell levels that fall within or above the range of B cellsand/or T cells of healthy, non-cancer patients. In some embodiments, theB cell and/or T cell levels per microliter of blood in the subject rangefrom about 225 to about 275 or more and from about 1350 to about 1650 ormore, respectively, e.g., when compared to healthy, non-cancer patients.

In some embodiments, methods for treating a subject having cancer withimmunotherapy are provided that comprise: (a) determining the number ofB cells and/or T cells in a subject with cancer; and (b) administeringto the subject an effective amount of an isolated binding molecule whichspecifically binds to semaphorin-4D (SEMA4D) and an effective amount ofat least one other immune modulating therapy if the number of B cellsand/or T cells in the subject exceeds a predetermined threshold level.In some embodiments, the predetermined threshold levels of B cellsand/or T cells per microliter of blood in the subject is about 1.5, 2,2.5, 3, 3.5, 4, 4.5, or 5 times the mean number of B cells and/or Tcells in circulation in other cancer patients. In some embodiments, thepredetermined threshold levels of B cells and/or T cells per microliterof blood in the subject range from about 147 to about 588 and from about1173 to about 3910, respectively, e.g., when compared to other cancerpatients. In some embodiments, the predetermined threshold levels of Bcells and/or T cells per microliter of blood in the subject fall withinor above the range of B cells and/or T cells of healthy, non-cancerpatients. In some embodiments, the predetermined threshold levels of Bcells and/or T cells per microliter of blood in the subject range fromabout 225 to about 275 or more and from about 1350 to about 1650, ormore, respectively, e.g., when compared to healthy, non-cancer patients.

In some embodiments, methods of treating a subject having cancer withimmunotherapy are provided that comprise: administering a combination ofan effective amount of an isolated binding molecule that specificallybinds to semaphorin-4D (SEMA4D) and an effective amount of at least oneother immune modulating therapy to a subject with cancer, whereinadministration of the combination results in enhanced therapeuticefficacy relative to administration of the isolated binding molecule orthe other immune modulating therapy alone. In some embodiments, theimmune modulating therapy is selected from the group consisting ofadministration of a cancer vaccine, administration of animmunostimulatory agent, adoptive T cell or antibody therapy,administration of an immune checkpoint blockade inhibitor,administration of a regulatory T cell (Treg) modulator, and acombination thereof In some embodiments, the immune modulating therapycomprises an immune checkpoint blockade inhibitor. In some embodiments,the immune checkpoint blockade inhibitor is an anti-CTLA4 antibody, ananti-PD-1 antibody, or a combination thereof. In some embodiments, theimmune modulating therapy comprises administration of a cancer vaccine.In some embodiments, the Treg modulator is cyclophosphamide. In someembodiments, the isolated binding molecule and the immune modulatingtherapy are administered separately or concurrently. In someembodiments, the subject has elevated levels of either B cells, T cellsor both B cells and T cells when compared to other cancer subjects. Insome embodiments, the levels of B cells and/or T cells per microliter ofblood in the subject is about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 timesthe mean number of B cells and/or T cells in circulation in other cancerpatients. In some embodiments, the levels of B cells and/or T cells permicroliter of blood in the subject range from about 147 to about 588 andfrom about 1173 to about 3910, respectively, e.g., when compared toother cancer patients. In some embodiments, the subject has levels of Bcells and/or T cells that fall within or above the range of B cellsand/or T cells of healthy, non-cancer patients. In some embodiments, thelevels of B cells and/or T cells per microliter of blood in the subjectrange from about 225 to about 275 or more and from about 1350 to about1650, or more, respectively, e.g., when compared to healthy, non-cancerpatients. In some embodiments, the cancer is selected from the groupconsisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer, gastriccancer, pancreatic cancer, neuroendocrine cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, brain cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial oruterine carcinoma, esophageal cancer, salivary gland carcinoma, kidneycancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer,head and neck cancer, and a combination thereof. In some embodiments ofany of the aforementioned methods, the isolated binding moleculespecifically binds to the same SEMA4D epitope as a reference monoclonalantibody selected from the group consisting of VX15/2503 or 67. In someembodiments of any of the aforementioned methods, the isolated bindingmolecule competitively inhibits a reference monoclonal antibody selectedfrom the group consisting of VX15/2503 or 67 from specifically bindingto SEMA4D. In some embodiments, the isolated binding molecule comprisesan antibody or antigen-binding fragment thereof. In some embodiments,the antibody or antigen-binding fragment thereof comprises the sixcomplementarity determining regions (CDRs) of monoclonal antibodyVX15/2503 or 67. In some embodiments, the antibody or antigen-bindingfragment thereof is monoclonal antibody VX15/2503 or 67.

Also provided are methods for inhibiting, delaying, or reducing growthof tumor cells expressing Her2 and Plexin B1, Plexin B2, or acombination thereof, comprising contacting the tumor cells with aneffective amount of an isolated binding molecule that specifically bindsto semaphorin-4D (SEMA4D), wherein growth of the tumor cells isinhibited, delayed, or reduced. In some embodiments, the contactingcomprises administration of the SEMA4D binding molecule to a subjectwith cancer, wherein the subject's cancer cells express Her2 and PlexinB1, Plexin B2, or a combination thereof. In some embodiments, the canceris breast cancer, ovarian cancer, lung cancer, or prostate cancer.

Also provided are methods for treating a subject having cancercomprising: (a) assaying the subject's cancer cells for expression ofHer2 and Plexin B1, Plexin B2, or a combination thereof and (b)administering to the subject an effective amount of an isolated bindingmolecule that specifically binds to semaphorin-4D (SEMA4D) if thesubject's cancer cells express Her2 and Plexin B1, Plexin B2, or acombination thereof. The method of claim 48 or claim 49, furthercomprising the administration of an effective amount of an anti-HER2/neubinding molecule. The method of claim 48 or claim 49, wherein theisolated binding molecule specifically binds to the same SEMA4D epitopeas a reference monoclonal antibody VX15/2503 or 67. The method of claim48 or claim 49, wherein the isolated binding molecule competitivelyinhibits a reference monoclonal antibody VX15/2503 or 67 fromspecifically binding to SEMA4D. The method of claim 48 or claim 49,wherein the isolated binding molecule comprises an antibody orantigen-binding fragment thereof. The method of claim 53, wherein theantibody or antigen-binding fragment thereof comprises a variable heavychain (VH) comprising VHCDRs 1-3 comprising SEQ ID NOs 6, 7, and 8,respectively, and a variable light chain (VL) comprising VLCDRs 1-3comprising SEQ ID NOs 14, 15, and 16, respectively. The method of claim54, wherein the VH and VL comprise, respectively, SEQ ID NO: 9 and SEQID NO: 17 or SEQ ID NO: 10 and SEQ ID NO: 18.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIGS. 1A-1B: Measurement of tumor volume in mice implanted withsyngeneic Colon26 tumor cells. FIG. 1A shows measurement of Colon26tumor volume in Balb/c and SCID mice treated twice weekly with either 1mg (50mg/kg) of anti-SEMA4D antibody (Ab) 67 or 2B8 isotype controlimmunoglobulin (2B8 Control Ig). FIG. 1B shows survival time, as definedin Example 1 below, of Balb/c and SCID mice treated with eitheranti-SEMA4D Ab 67 or 2B8 Control Ig.

FIG. 2: Shows measurement of Colon26 tumor volume in Balb/c miceimplanted with tumor cells and treated first with anti-CD8 depletingantibody (Clone 2.43, BioXCell) or control Rat Ig (150 mg/kg) and thentreated as in FIG. 1A with either 2B8 Control Ig or anti-SEMA4D Ab 67.

FIGS. 3A-3B: Measurement of immune cell density in Colon26 tumor ofgrafted mice.

FIG. 3A shows density of CD8+ T cells as determined by % tumor areastained with anti-CD8 antibody after treatment with Control Ig oranti-SEMA4D Ab 67. FIG. 3B shows density of CD20+ B cells as determinedby % tumor area stained with anti-CD20 antibody after treatment withControl Ig or anti-SEMA4D Ab 67.

FIGS. 4A-4D: Measurement of macrophage and CD8+ T cell distribution atleading edge of tumor in Colon26 grafted mice. FIG. 4A shows images ofrepresentative Colon26 tumors from mice grafted 27 days earlier andtreated with either Control Ig or anti-SEMA4D Ab 67 as described inFIG. 1. FIG. 4B shows measurement of M1 type macrophage density atleading edge of tumor, defined as a 300 pixel wide region (250 micron)from the edge of the tumor, as determined by % pixel area stained withanti-F4/80 antibody. FIG. 4C shows measurement of M2 type macrophagedensity at leading edge of tumor as determined by % pixel area stainedwith anti-CD206 antibody. FIG. 4D shows measurement of CD8+T celldensity at leading edge of tumor, as determined by % pixel area stainedwith cytotoxic T cell anti-CD8 antibody.

FIGS. 5A-5D: Measurement of tumor volume in mice implanted withsyngeneic Colon26 tumor cells. FIG. 5A shows measurement of Colon26tumor volume in Balb/c mice treated with either control Mouse IgG1/2B8or anti-SEMA4D/MAb 67-2 (50 mg/kg, IP, weekly), with or withoutanti-CTLA4/MAb UC10-4F10-11 (100 μg on day 8 and 50 μg on days 11 and 14post tumor inoculation), and with anti-PD1/RMP1-14 (100 μg on day 3,twice weekly) in combination with anti-CTLA4/MAb UC10-4F10-11. FIG. 5Bshows survival time of Balb/c mice treated with either control MouseIgG1/2B8 or anti-SEMA4D/MAb 67-2, with or without anti-CTLA4/MAbUC10-4F10-11, and with anti-PD1/RMP1-14 (100 μg on day 3, twice weekly)in combination with anti-CTLA4/MAb UC10-4F10-11. FIG. 5C shows thefrequency of tumor regression in Balb/c mice treated with either controlMouse IgG1/2B8 or anti-SEMA4D/MAb 67-2, with or without anti-CTLA4/MAbUC10-4F10-11, and with anti-PD1/RMP1-14 (100 μg on day 3, twice weekly)in combination with anti-CTLA4/MAb UC10-4F10-11 (p values, *0.05 and**0.01). FIG. 5D shows measurements of pro-inflammatory cytokines IFNγin the tumor infiltrating lymphocytes of mice treated with thecombination of anti-SEMA4D/MAb 67-2 and anti-CTLA4/MAb UC10-4F10-11compared to either control Mouse IgG1/2B8 or monotherapy (eitheranti-SEMA4D/MAb 67-2 or anti-CTLA4/MAb UC10-4F10-11). FIG. 5E showsfrequency of peptide-specific IFNγ secreting responders among tumorinfiltrating lymphocytes recovered from spleen of mice treated with thecombination of anti-SEMA4D/MAb 67-2 and anti-CTLA4/MAb UC10-4F10-11compared to either control Mouse IgG1/2B8 or monotherapy (eitheranti-SEMA4D/MAb 67-2 or anti-CTLA4/MAb UC10-4F10-11).

FIGS. 6A-6E: Measurement of an anti-SEMA4D antibody to affect tumorinfiltration of tumor-specific cytotoxic CD8+ T cells. FIG. 6A showsmeasurement of IFNγ secreting cells in MAb 67-treated mice both in thepresence and absence of peptide. FIG. 6B shows representative ELISPOTimages. FIG. 6C shows measurement of anti-tumor cytokines, such as IFNγand TNFα, in tumor-infiltrating lymphocytes (TIL). FIG. 6D showsmeasurements of pro-inflammatory cytokines IFNγ and TNFα in the TIL ofmice treated with the anti-SEMA4D/MAb 67 antibody. FIG. 6E showsfrequency of peptide-specific IFNγ secreting responders in the tumorinfiltrating lymphocytes of mice treated with anti-SEMA4D/MAb 67antibody.

FIGS. 7A-7D: Measurement of tumor volume in mice implanted withsyngeneic Colon26 tumor cells. FIG. 7A shows measurement of Colon26tumor volume in Balb/c mice treated with either control Mouse IgG1/2B8or anti-SEMA4D/MAb 67-2 (50 mg/kg, IP, weekly) together with eithercontrol rat Ig or rat anti-PD1/MAbRMP1-14 (100 μg, twice per week, for 2weeks starting at 3 days post tumor inoculation). FIG. 7B shows survivaltime of Balb/c mice treated with either control Mouse IgG1/2B8 oranti-SEMA4D/MAb 67-2 together with either control rat Ig or ratanti-PD1/MAbRMP1-14. FIGS. 7C and 7D show the frequency of tumorregression in Balb/c mice treated with either control Mouse IgG1/2B8 oranti-SEMA4D/MAb 67-2 together with either control rat Ig or ratanti-PD1/MAbRMP1-14.

FIGS. 8A-8E: Measurement of tumor volume in mice implanted withsyngeneic Colon26 tumor cells. FIG. 8A shows mean measurement of Colon26tumor volume in Balb/c mice treated with either control Mouse IgG1/2B8or anti-SEMA4D/MAb 67-2 (50 mg/kg, IP, weekly), with or withoutcyclophosphamide (CY) (50 mg/kg, IP). FIG. 8B shows median measurementof Colon26 tumor volume in Balb/c mice treated with either control MouseIgG1/2B8 or anti-SEMA4D/MAb 67-2 (50 mg/kg, IP, weekly), with or withoutcyclophosphamide (CY) (50 mg/kg, IP). FIG. 8C shows survival time ofBalb/c mice treated with either control Mouse IgG1/2B8 oranti-SEMA4D/MAb 67-2, with or without cyclophosphamide. FIGS. 8D and 8Eshow the frequency of tumor regressions in Balb/c mice treated witheither control Mouse IgG1/2B8 or anti-SEMA4D/MAb 67-2, with or withoutcyclophosphamide (CY).

FIGS. 9A-9C: Measurement of tumor volume in mice implanted with Tubo.A5tumor cells. FIG. 9A shows measurement of tumor volume in Balb/c micetreated with either control Mouse IgG1/2B8 or anti-SEMA4D/MAb 67-2 (50mg/kg, IP, weekly), with or without anti-Neu/MAb7.16.4 (αNeu) (200μg IPweekly ×2 starting when Tumor Volume (TV) is approximately 200 mm³, ondays 21 and 28). FIG. 9B shows survival time of Balb/c mice treated witheither control Mouse IgG1/2B8 or anti-SEMA4D/MAb 67-2, with or withoutanti-Neu/MAb7.16.4 (αNeu). FIG. 9C shows the frequency of tumorregressions in Balb/c mice treated with either control Mouse IgG1/2B8 oranti-SEMA4D/MAb 67-2, with or without anti-Neu/MAb7.16.4 (αNeu).

FIGS. 10A-10E: Measurement of tumor volume in Balb/c mice implanted withTubo.A5 tumor cells. FIG. 10A shows measurement of tumor volume inBalb/c mice treated with either control Mouse IgG1/2B8 oranti-SEMA4D/MAb 67-2 (50 mg/kg, IP, weekly). FIG. 10B shows survivaltime of Balb/c mice treated with either control Mouse IgG1/2B8 oranti-SEMA4D/MAb 67-2. FIGS. 10C-10E show the frequency of tumorregressions in the Tubo.A5 tumor model. Specifically, FIG. 10C showscontrol mice grafted with the Tubo.A5 tumor. FIG. 10D shows mice thathave rejected Tubo.A5 tumor grafts following treatment withanti-SEMA4D/MAb 67-2 and that were rechallenged with Tubo.A5 tumor onday 90 following the original graft. FIG. 10E shows naïve micechallenged with the same tumor graft as in FIG. 10D to demonstrate tumorviability in vivo.

FIGS. 11A-11B: Measurement of T cell infiltration and MDSC in Tubo.A5tumor models. FIG. 11A shows measurement of CD3+ T cells in tumors ofBalb/c mice treated with either control Mouse IgG1/2B8 oranti-SEMA4D/MAb 67-2 (50 mg/kg, IP, weekly). FIG. 11B shows measurementof CD11b+Gr1+ MDSC in tumors of Balb/c mice treated with either controlMouse IgG1/2B8 or anti-SEMA4D/MAb 67-2 (50 mg/kg, IP, weekly).

FIGS. 12A-12D: Measurement of tumor volume in mice implanted with eitherColon 26 or Tubo.A5 tumor cells. FIG. 12A shows measurement of Tubo.A5tumor volume in Balb/c mice treated with either control MouseIgG1/2B8.1E7 (50 mg/kg, IP, weekly ×6) or varying levels ofanti-SEMA4D/MAb 67-2 (1, 10 or 50 mg/kg, IP, weekly ×6). FIG. 12B showssurvival time of Balb/c mice treated with either control MouseIgG1/2B8.IE7 (50 mg/kg, IP, weekly ×6) or varying levels ofanti-SEMA4D/MAb 67-2 (1, 10 or 50 mg/kg, IP, weekly ×6). FIG. 12C showsmeasurement of Colon 26 tumor volume in Balb/c mice treated with controlMouse IgG1/2B8.IE7 (50 mg/kg, IP, weekly ×5), anti-SEMA4D/MAb 67-2 (50mg/kg, IP, weekly ×5), anti-CTLA4/MAb UC10-4F10-11 (5mg/kg, IP, weekly×5), or a combination of anti-CTLA4/MAb UC10-4F10-11 (5mg/kg, IP, weekly×5) and varying levels of anti-SEMA4D/MAb 67-2 (0.3, 3, 10, or 50 mg/kg,IP, weekly ×5). FIG. 12D shows survival time of Balb/c mice treated withcontrol Mouse IgG1/2B8.IE7 (50 mg/kg, IP, weekly ×5), anti-SEMA4D/MAb67-2 (50 mg/kg, IP, weekly ×5), anti-CTLA4/MAb UC10-4F10-11 (5mg/kg, IP,weekly ×5), or a combination of anti-CTLA4/MAb UC10-4F10-11 (5mg/kg, IP,weekly ×5) and varying levels of anti-SEMA4D/MAb 67-2 (0.3, 3, 10, or 50mg/kg, IP, weekly ×5).

FIG. 13: Summary of experiments conducted in above figures showing tumorregressions and growth after tumor re-challenge in Colon26 and Tubo.A5tumor models.

DETAILED DESCRIPTION Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a polynucleotide,” is understood torepresent one or more polynucleotides. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term and/or” as used in a phrase such as “Aand/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects orembodiments of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

Wherever embodiments are described with the language “comprising,”otherwise analogous embodiments described in terms of “consisting ofand/or “consisting essentially of” are also provided.

Amino acids are referred to herein by their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter codes.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer, gastric,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, brain cancer, hepatoma, breast cancer, coloncancer, colorectal cancer, endometrial or uterine carcinoma, esophagealcancer, salivary gland carcinoma, sarcoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma andvarious types of head and neck cancers.

In certain embodiments, metastatic cancers that are amenable totreatment via the methods provided herein include, but are not limitedto metastatic sarcomas, breast carcinomas, ovarian cancer, head and neckcancer, and pancreatic cancer. In certain embodiments metastatic cancersor tumor cells that are amenable to treatment via the methods providedherein express Plexin-B1 and/or Plexin-B2 receptors for SEMA4D.

“Angiogenesis” refers to a complex multistep morphogenetic event duringwhich endothelial cells, stimulated by major determinants of vascularremodeling, dynamically modify their cell-to-cell and cell-to-matrixcontacts and move directionally to be reorganized into a mature vasculartree (Bussolino et al., Trends Biochem Sci. 22:251-256 (1997); Risau,Nature 386:671-674 (1997); Jain, Nat. Med. 9:685-693 (2003)). Theformation of new blood vessels is a key step during embryo development,but it also occurs in adults in physiologic and in pathologicconditions, such as retinopathy, rheumatoid arthritis, ischemia, andparticularly tumor growth and metastasis (Carmeliet, Nat. Med. 9:653-660(2003)).

As used herein, the term “clinical laboratory” refers to a facility forthe examination or processing of materials derived from a livingsubject, e.g., a human being. Non-limiting examples of processinginclude biological, biochemical, serological, chemical,immunohematological, hematological, biophysical, cytological,pathological, genetic, or other examination of materials derived fromthe human body for the purpose of providing information, e.g., for thediagnosis, prevention, or treatment of any disease or impairment of, orthe assessment of the health of living subjects, e.g., human beings.These examinations can also include procedures to collect or otherwiseobtain a sample, prepare, determine, measure, or otherwise describe thepresence or absence of various substances in the body of a livingsubject, e.g., a human being, or a sample obtained from the body of aliving subject, e.g., a human being.

The terms “proliferative disorder” and “proliferative disease” refer todisorders associated with abnormal cell proliferation such as cancer.

“Tumor” and “neoplasm” as used herein refer to any mass of tissue thatresult from excessive cell growth or proliferation, either benign(noncancerous) or malignant (cancerous) including pre-cancerous lesions.In certain embodiments, tumors described herein express Plexin-B1 and/orPlexin-B2, and can express SEMA4D and activated Met.

As used herein, the term “healthcare benefits provider” encompassesindividual parties, organizations, or groups providing, presenting,offering, paying for in whole or in part, or being otherwise associatedwith giving a patient access to one or more healthcare benefits, benefitplans, health insurance, and/or healthcare expense account programs.

The term “immune modulating therapy” or “immunotherapy” refers totreatment that impacts a disease or disorder in a subject by inducingand/or enhancing an immune response in that subject. Immune modulatingtherapies include cancer vaccines, immunostimulatory agents, adoptive Tcell or antibody therapy, and immune checkpoint blockade (Lizée et al.2013. Harnessing the Power of the Immune System to Target Cancer. Annu.Rev. Med. Vol. 64 No. 71-90).

The term “immune modulating agent” refers to the active agents ofimmunotherapy. Immune modulating agents include a diverse array ofrecombinant, synthetic and natural, preparation. Examples of immunemodulating agents include, but are not limited to, interleukins such asIL-2, IL-7, IL-12; cytokines such as granulocyte colony-stimulatingfactor (G-CSF), interferons; various chemokines such as CXCL13, CCL26,CXCL7; antagonists of immune checkpoint blockades such as anti-CTLA-4,anti-PD1 or anti-PD-L1 (ligand of PD-1), anti-LAG3, anti-B7-H3,synthetic cytosine phosphate-guanosine (CpG) oligodeoxynucleotides,glucans; and modulators of regulatory T cells (Tregs) such ascyclophosphamide.

The terms “metastasis,” “metastases,” “metastatic,” and othergrammatical equivalents as used herein refer to cancer cells whichspread or transfer from the site of origin (e.g., a primary tumor) toother regions of the body with the development of a similar cancerouslesion at the new location. A “metastatic” or “metastasizing” cell isone that loses adhesive contacts with neighboring cells and migrates viathe bloodstream or lymph from the primary site of disease to invadeneighboring body structures. The terms also refer to the process ofmetastasis, which includes, but is not limited to detachment of cancercells from a primary tumor, intravasation of the tumor cells tocirculation, their survival and migration to a distant site, attachmentand extravasation into a new site from the circulation, andmicrocolonization at the distant site, and tumor growth and developmentat the distant site.

The term “therapeutically effective amount” refers to an amount of anantibody, polypeptide, polynucleotide, small organic molecule, or otherdrug effective to “treat” a disease or disorder in a subject or mammal.In the case of cancer, the therapeutically effective amount of the drugcan reduce the number of cancer cells; retard or stop cancer celldivision, reduce or retard an increase in tumor size; inhibit, e.g.,suppress, retard, prevent, stop, delay, or reverse cancer cellinfiltration into peripheral organs including, for example, the spreadof cancer into soft tissue and bone; inhibit, e.g., suppress, retard,prevent, shrink, stop, delay, or reverse tumor metastasis; inhibit,e.g., suppress, retard, prevent, stop, delay, or reverse tumor growth;relieve to some extent one or more of the symptoms associated with thecancer, reduce morbidity and mortality; improve quality of life; or acombination of such effects. To the extent the drug prevents growthand/or kills existing cancer cells, it can be referred to as cytostaticand/or cytotoxic.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, reverse, and/or halt progression of adiagnosed pathologic condition or disorder and 2) prophylactic orpreventative measures that prevent and/or slow the development of atargeted pathologic condition or disorder. Thus those in need oftreatment include those already with the disorder; those prone to havethe disorder; and those in whom the disorder is to be prevented. Asubject is successfully “treated” according to the methods of thepresent disclosure if the patient shows one or more of the following: areduction in the number of or complete absence of cancer cells; areduction in the tumor size; or retardation or reversal of tumor growth,inhibition, e.g., suppression, prevention, retardation, shrinkage,delay, or reversal of metastases, e.g., of cancer cell infiltration intoperipheral organs including, for example, the spread of cancer into softtissue and bone; inhibition of, e.g., suppression of, retardation of,prevention of, shrinkage of, reversal of, delay of, or an absence oftumor metastases; inhibition of, e.g., suppression of, retardation of,prevention of, shrinkage of, reversal of, delay of, or an absence oftumor growth; relief of one or more symptoms associated with thespecific cancer; reduced morbidity and mortality; improvement in qualityof life; or some combination of effects. Beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already with thecondition or disorder as well as those prone to have the condition ordisorder or those in which the condition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows,bears, and so on.

As used herein, phrases such as “a subject that would benefit fromadministration of an anti-SEMA4D antibody as a single agent or incombination with at least one other immune modulating therapy” and “ananimal in need of treatment” includes subjects, such as mammaliansubjects, that would benefit from administration of an anti-SEMA4Dantibody as a single agent or in combination with at least one otherimmune modulating therapy.

A “binding molecule” or “antigen binding molecule” of the presentdisclosure refers in its broadest sense to a molecule that specificallybinds an antigenic determinant. In one embodiment, the binding moleculespecifically binds to SEMA4D, e.g., a transmembrane SEMA4D polypeptideof about 150 kDa or a soluble SEMA4D polypeptide of about 120 kDa(commonly referred to as sSEMA4D). In another embodiment, a bindingmolecule of the disclosure is an antibody or an antigen binding fragmentthereof. In another embodiment, a binding molecule of the disclosurecomprises at least one heavy or light chain Complementarity DeterminingRegion (CDR) of an antibody molecule. In another embodiment, a bindingmolecule of the disclosure comprises at least two CDRs from one or moreantibody molecules. In another embodiment, a binding molecule of thedisclosure comprises at least three CDRs from one or more antibodymolecules. In another embodiment, a binding molecule of the disclosurecomprises at least four CDRs from one or more antibody molecules. Inanother embodiment, a binding molecule of the disclosure comprises atleast five CDRs from one or more antibody molecules. In anotherembodiment, a binding molecule of the disclosure comprises at least sixCDRs from one or more antibody molecules. In another embodiment, thebinding molecule can be an antagonist of the Plexin-B1 receptor forSEMA4D. By antagonist is meant a binding molecule that interferes withthe signaling function of the receptor. The antagonist can competitivelyblock binding of a natural ligand but fail to trigger the normalphysiological response. Binding molecules can be antibodies or antigenbinding fragments thereof as described above or can be other biologicsor small molecule drugs that act as competitive inhibitors or interferewith signaling by natural ligands. The present disclosure is directed toa method of inhibiting tumor growth and metastases in a subject, e.g.,cancer patient, comprising administering to the subject an anti-SEMA4Dbinding molecule, e.g., an antibody, or antigen-binding fragment,variant, or derivative thereof, as a single agent or in combination withat least one other immune modulating therapy. Unless specificallyreferring to full-sized antibodies such as naturally occurringantibodies, the term “anti-SEMA4D antibody” encompasses full-sizedantibodies as well as antigen-binding fragments, variants, analogs, orderivatives of such antibodies, e.g., naturally occurring antibody orimmunoglobulin molecules or engineered antibody molecules or fragmentsthat bind antigen in a manner similar to antibody molecules. Alsoincluded in SEMA4D binding molecules are other biologics or smallmolecules that bind and inhibit the activity of SEMA4D or of itsPlexin-B1 receptor.

As used herein, “human” or “fully human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins, as described infraand, for example, in U.S. Pat. No. 5,939,598 by Kucherlapati et al.“Human” or “fully human” antibodies also include antibodies comprisingat least the variable domain of a heavy chain, or at least the variabledomains of a heavy chain and a light chain, where the variable domain(s)have the amino acid sequence of human immunoglobulin variable domain(s).

“Human” or “fully human” antibodies also include “human” or “fullyhuman” antibodies, as described above, that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the VH regions and/or VL regions) describedherein, which antibodies or fragments thereof immunospecifically bind toa SEMA4D polypeptide or fragment or variant thereof. Standard techniquesknown to those of skill in the art can be used to introduce mutations inthe nucleotide sequence encoding a human anti-SEMA4D antibody,including, but not limited to, site-directed mutagenesis andPCR-mediated mutagenesis which result in amino acid substitutions. Incertain aspects, the variants (including derivatives) encode less than50 amino acid substitutions, less than 40 amino acid substitutions, lessthan 30 amino acid substitutions, less than 25 amino acid substitutions,less than 20 amino acid substitutions, less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the reference VH region, VHCDR1, VHCDR2, VHCDR3, VL region,VLCDR1, VLCDR2, or VLCDR3.

In certain embodiments, the amino acid substitutions are conservativeamino acid substitution, discussed further below. Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity (e.g., the ability to bind a SEMA4D polypeptide, e.g., human,murine, or both human and murine SEMA4D). Such variants (or derivativesthereof) of “human” or “fully human” antibodies can also be referred toas human or fully human antibodies that are “optimized” or “optimizedfor antigen binding” and include antibodies that have improved affinityto antigen.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory Press).

As used herein, the term “immunoglobulin” comprises various broadclasses of polypeptides that can be distinguished biochemically. Thoseskilled in the art will appreciate that heavy chains are classified asgamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, γ) with somesubclasses among them (e.g., γ1-γ4). It is the nature of this chain thatdetermines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE,respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2,IgG3, IgG4, IgA1, etc. are well characterized and are known to conferfunctional specialization. Modified versions of each of these classesand isotypes are readily discernable to the skilled artisan in view ofthe instant disclosure and, accordingly, are within the scope of theinstant disclosure. All immunoglobulin classes are clearly within thescope of the present disclosure, the following discussion will generallybe directed to the IgG class of immunoglobulin molecules. With regard toIgG, a standard immunoglobulin molecule comprises two identical lightchain polypeptides of molecular weight approximately 23,000 Daltons, andtwo identical heavy chain polypeptides of molecular weight53,000-70,000. The four chains are typically joined by disulfide bondsin a “Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class can be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL or VK) and heavy (VH) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3)confer important biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs) within these variable domains, of an antibodycombine to form the variable region that defines a three dimensionalantigen binding site. This quaternary antibody structure forms theantigen binding site present at the end of each arm of the Y. Morespecifically, the antigen binding site is defined by three CDRs on eachof the VH and VL chains. In some instances, e.g., certain immunoglobulinmolecules derived from camelid species or engineered based on camelidimmunoglobulins, a complete immunoglobulin molecule can consist of heavychains only, with no light chains. See, e.g., Hamers-Casterman et al.,Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops that connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable domainby one of ordinary skill in the art, since they have been preciselydefined (see below).

In the case where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al. (1983) U.S. Dept. of Health and HumanServices, “Sequences of Proteins of Immunological Interest” and byChothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which areincorporated herein by reference, where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. The appropriate amino acid residuesthat encompass the CDRs as defined by each of the above cited referencesare set forth below in Table 1 as a comparison. The exact residuenumbers that encompass a particular CDR will vary depending on thesequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular CDR given the variableregion amino acid sequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia VH CDR1 31-35  26-32  VH CDR250-65  52-58  VH CDR3 95-102 95-102 VL CDR1 24-34  26-32  VL CDR2 50-56 50-52  VL CDR3 89-97  91-96  ¹Numbering of all CDR definitions in Table1 is according to the numbering conventions set forth by Kabat et at.(see below).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al. (1983) U.S. Dept. ofHealth and Human Services, “Sequence of Proteins of ImmunologicalInterest.” Unless otherwise specified, references to the numbering ofspecific amino acid residue positions in an anti-SEMA4D antibody orantigen-binding fragment, variant, or derivative thereof of the presentdisclosure are according to the Kabat numbering system.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the disclosure include, but are not limited to, polyclonal,monoclonal, multispecific, bispecific, human, humanized, primatized, orchimeric antibodies, single-chain antibodies, epitope-binding fragments,e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv),disulfide-linked Fvs (sdFv), fragments comprising either a VL or VHdomain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto anti-SEMA4D antibodies disclosed herein). ScFv molecules are known inthe art and are described, e.g., in U.S. Pat. No. 5,892,019.Immunoglobulin or antibody molecules of the disclosure can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2, etc.), or subclass of immunoglobulinmolecule.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. In certainembodiments, a polypeptide comprising a heavy chain portion comprises atleast one of: a VH domain, a CH1 domain, a hinge (e.g., upper, middle,and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or avariant or fragment thereof. For example, a binding polypeptide for usein the disclosure can comprise a polypeptide chain comprising a CH1domain; a polypeptide chain comprising a CH1 domain, at least a portionof a hinge domain, and a CH2 domain; a polypeptide chain comprising aCH1 domain and a CH3 domain; a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, and a CH3 domain, or apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, a CH2 domain, and a CH3 domain. In another embodiment, apolypeptide of the disclosure comprises a polypeptide chain comprising aCH3 domain. Further, a binding polypeptide for use in the disclosure canlack at least a portion of a CH2 domain (e.g., all or part of a CH2domain). As set forth above, it will be understood by one of ordinaryskill in the art that these domains (e.g., the heavy chain portions) canbe modified such that they vary in amino acid sequence from thenaturally occurring immunoglobulin molecule.

In certain anti-SEMA4D antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein, the heavy chainportions of one polypeptide chain of a multimer are identical to thoseon a second polypeptide chain of the multimer. Alternatively, heavychain portion-containing monomers of the disclosure are not identical.For example, each monomer can comprise a different target binding site,forming, for example, a bispecific antibody. A bispecific antibody is anartificial protein that is composed of fragments of two differentmonoclonal antibodies and consequently binds to two different types ofantigen. Variations on the bispecific antibody format are contemplatedwithin the scope of the present disclosure. Bispecific antibodies can begenerated using techniques that are well known in the art for example,see, for example, Ghayur et al., Expert Review of Clinical Pharmacology3.4 (July 2010): p491; Lu et al., J. Biological Chemistry Vol. 280, No.20, p. 19665-19672 (2005); Marvin et al., Acta Pharmacologic Sinica26(6):649-658 (2005); and Milstein C et al., Nature 1983; 305: 537-40;30 Brennan M et al., Science 1985; 229: 81-3; Thakur et al., Curr OpinMol Ther. 2010 June; 12(3):340-9; and U.S. Patent Publication No.2007/0004909.

The heavy chain portions of a binding molecule for use in the methodsdisclosed herein can be derived from different immunoglobulin molecules.For example, a heavy chain portion of a polypeptide can comprise a CH1domain derived from an IgG1 molecule and a hinge region derived from anIgG3 molecule. In another example, a heavy chain portion can comprise ahinge region derived, in part, from an IgG1 molecule and, in part, froman IgG3 molecule. In another example, a heavy chain portion can comprisea chimeric hinge derived, in part, from an IgG1 molecule and, in part,from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain, e.g., a kappa orlambda light chain. In certain aspects, the light chain portioncomprises at least one of a VL or CL domain.

Anti-SEMA4D antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein can be described or specified interms of the epitope(s) or portion(s) of an antigen, e.g., a targetpolypeptide disclosed herein (e.g., SEMA4D) that they recognize orspecifically bind. The portion of a target polypeptide that specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant.” A target polypeptide cancomprise a single epitope, but typically comprises at least twoepitopes, and can include any number of epitopes, depending on the size,conformation, and type of antigen. Furthermore, it should be noted thatan “epitope” on a target polypeptide can be or can includenon-polypeptide elements, e.g., an epitope can include a carbohydrateside chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes can contain at least seven, at least nine and, in some cases,between at least about 15 to about 30 amino acids. Since a CDR canrecognize an antigenic peptide or polypeptide in its tertiary form, theamino acids comprising an epitope need not be contiguous, and in somecases, may not even be on the same peptide chain. A peptide orpolypeptide epitope recognized by anti-SEMA4D antibodies of the presentdisclosure can contain a sequence of at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 15, at least20, at least 25, or between about 15 to about 30 contiguous ornon-contiguous amino acids of SEMA4D.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” can be deemed to have a higher specificity or affinity fora given epitope than antibody “B,” or antibody “A” can be said to bindto epitope “C” with a higher specificity or affinity than it has forrelated epitope “D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody that“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody cancross-react with the related epitope.

By way of non-limiting example, an antibody can be considered to bind afirst epitope preferentially if it binds said first epitope with adissociation constant (KD) that is less than the antibody's KD for thesecond epitope. In another non-limiting example, an antibody can beconsidered to bind a first antigen preferentially if it binds the firstepitope with an affinity that is at least one order of magnitude lessthan the antibody's KD for the second epitope. In another non-limitingexample, an antibody can be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's KD for the secondepitope.

In another non-limiting example, an antibody can be considered to bind afirst epitope preferentially if it binds the first epitope with an offrate (k(off)) that is less than the antibody's k(off) for the secondepitope. In another non-limiting example, an antibody can be consideredto bind a first epitope preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope. In another non-limitingexample, an antibody can be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's k(off) for thesecond epitope.

An antibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition can be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody can be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of animmunoglobulin molecule. See, e.g., Harlow et al. (1988) Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) pages27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity.

Anti-SEMA4D antibodies or antigen-binding fragments, variants, orderivatives thereof of the disclosure can also be described or specifiedin terms of their cross-reactivity. As used herein, the term“cross-reactivity” refers to the ability of an antibody, specific forone antigen, to react with a second antigen; a measure of relatednessbetween two different antigenic substances. Thus, an antibody is crossreactive if it binds to an epitope other than the one that induced itsformation. The cross reactive epitope generally contains many of thesame complementary structural features as the inducing epitope, and insome cases, can actually fit better than the original.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody can be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody can be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Anti-SEMA4D binding molecules, e.g., antibodies or antigen-bindingfragments, variants or derivatives thereof of the disclosure can also bedescribed or specified in terms of their binding affinity to apolypeptide of the disclosure, e.g., SEMA4D, e.g., human, murine, orboth human and murine SEMA4D. In certain aspects, binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻²M,10⁻²M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴M, 10⁻⁴M, 5×10⁻⁵M, 10⁻⁵M, 5×10⁻⁶ M, 10⁶M,5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M,10⁻¹⁴M, 5×10⁻¹⁵M, or 10⁻¹⁵M.

In certain embodiments, the anti-SEMA4D binding molecule, e.g., anantibody or antigen binding fragment thereof, of the disclosure bindshuman SEMA4D with a Kd of about 5×10⁻⁹ to about 6×10⁻⁹. In anotherembodiment, the anti-SEMA4D binding molecule, e.g., an antibody orantigen binding fragment thereof, of the disclosure binds murine SEMA4Dwith a Kd of about 1×10⁻⁹ to about 2×10⁻⁹.

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which can beintact, partial or modified) is obtained from a second species. In someembodiments the target binding region or site will be from a non-humansource (e.g., mouse or primate) and the constant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy or light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs can bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class orfrom an antibody from a different species. An engineered antibody inwhich one or more “donor” CDRs from a non-human antibody of knownspecificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” In certainaspects it is not necessary to replace all of the CDRs with the completeCDRs from the donor variable domain to transfer the antigen bindingcapacity of one variable domain to another. Rather, only those residuesthat are necessary to maintain the activity of the binding site againstthe targeted antigen can be transferred.

It is further recognized that the framework regions within the variabledomain in a heavy or light chain, or both, of a humanized antibody cancomprise solely residues of human origin, in which case these frameworkregions of the humanized antibody are referred to as “fully humanframework regions” (for example, MAb VX15/2503, disclosed in U.S. PatentAppl. Publication No. US 2010/0285036 A1 as MAb 2503, incorporatedherein by reference in its entirety). Alternatively, one or moreresidues of the framework region(s) of the donor variable domain can beengineered within the corresponding position of the human frameworkregion(s) of a variable domain in a heavy or light chain, or both, of ahumanized antibody if necessary to maintain proper binding or to enhancebinding to the SEMA4D antigen. A human framework region that has beenengineered in this manner would thus comprise a mixture of human anddonor framework residues, and is referred to herein as a “partiallyhuman framework region.”

For example, humanization of an anti-SEMA4D antibody can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodentor mutant rodent CDRs or CDR sequences for the corresponding sequencesof a human anti-SEMA4D antibody. See also U.S. Pat. Nos. 5,225,539;5,585,089; 5,693,761; 5,693,762; 5,859,205; herein incorporated byreference. The resulting humanized anti-SEMA4D antibody would compriseat least one rodent or mutant rodent CDR within the fully humanframework regions of the variable domain of the heavy and/or light chainof the humanized antibody. In some instances, residues within theframework regions of one or more variable domains of the humanizedanti-SEMA4D antibody are replaced by corresponding non-human (forexample, rodent) residues (see, for example, U.S. Pat. Nos. 5,585,089;5,693,761; 5,693,762; and 6,180,370), in which case the resultinghumanized anti-SEMA4D antibody would comprise partially human frameworkregions within the variable domain of the heavy and/or light chain.Similar methods can be used for humanization of an anti-VEGF antibody.

Furthermore, humanized antibodies can comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance (e.g., toobtain desired affinity). In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDRs correspond tothose of a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature 331:522-525(1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992); herein incorporated by reference.Accordingly, such “humanized” antibodies can include antibodies whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some framework residues are substitutedby residues from analogous sites in rodent antibodies. See, for example,U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205.See also U.S. Pat. No. 6,180,370, and International Publication No. WO01/27160, where humanized antibodies and techniques for producinghumanized antibodies having improved affinity for a predeterminedantigen are disclosed.

Target Polypeptide Description—SEMA4D

As used herein, the terms “semaphorin-4D”, “SEMA4D”, and “SEMA4Dpolypeptide” are used interchangeably, as are “SEMA4D” and “Sema4D.” Incertain embodiments, SEMA4D is expressed on the surface of or secretedby a cell. In another embodiment, SEMA4D is membrane bound. In anotherembodiment, SEMA4D is soluble, e.g., sSEMA4D. In another embodiment,SEMA4D can include a full-sized SEMA4D or a fragment thereof, or aSEMA4D variant polypeptide, wherein the fragment of SEMA4D or SEMA4Dvariant polypeptide retains some or all functional properties of thefull-sized SEMA4D.

The full-sized human SEMA4D protein is a homodimeric transmembraneprotein consisting of two polypeptide chains of 150 kDa. SEMA4D belongsto the semaphorin family of cell surface receptors and is also referredto as CD100. Both human and mouse SEMA4D/Sema4D are proteolyticallycleaved from their transmembrane form to generate 120-kDa soluble forms,giving rise to two Sema4D isoforms (Kumanogoh et al., J. Cell Science116(7):3464 (2003)). Semaphorins consist of soluble and membrane-boundproteins that were originally defined as axonal-guidance factors whichplay an important role in establishing precise connections betweenneurons and their appropriate target. Structurally considered a class IVsemaphorin, SEMA4D consists of an amino-terminal signal sequencefollowed by a characteristic ‘Sema’ domain, which contains 17 conservedcysteine residues, an Ig-like domain, a lysine-rich stretch, ahydrophobic transmembrane region, and a cytoplasmic tail.

The SEMA4D polypeptide includes a signal sequence of about 13 aminoacids followed by a semaphorin domain of about 512 amino acids, animmunoglobulin-like (Ig-like) domain of about 65 amino acids, alysine-rich stretch of 104 amino acids, a hydrophobic transmembraneregion of about 19 amino acids, and a cytoplasmic tail of 110 aminoacids. A consensus site for tyrosine phosphorylation in the cytoplasmictail supports the predicted association of SEMA4D with a tyrosine kinase(Schlossman et al., Eds. (1995) Leucocyte Typing V (Oxford UniversityPress, Oxford).

SEMA4D is known to have at least three functional receptors, Plexin-B1,Plexin-B2 and CD72. Plexin-B1, is expressed in non-lymphoid tissues andhas been shown to be a high affinity (1 nM) receptor for SEMA4D(Tamagnone et al., Cell 99:71-80 (1999)). SEMA4D stimulation of PlexinB1 signaling has been shown to induce growth cone collapse of neurons,and to induce process extension collapse and apoptosis ofoligodendrocytes (Giraudon et al., J. Immunol. 172:1246-1255 (2004);Giraudon et al., NeuroMolecular Med. 7:207-216 (2005)). After binding toSEMA4D, Plexin B1 signaling mediates the inactivation of R-Ras, leadingto a decrease in the integrin mediated attachment to the extracellularmatrix, as well as to activation of RhoA, leading to cell collapse byreorganization of the cytoskeleton. See Kruger et al., Nature Rev. Mol.Cell Biol. 6:789-800 (2005); Pasterkamp, TRENDS in Cell Biology 15:61-64(2005)). Plexin-B2 has an intermediate affinity for SEMA4D and a recentreport indicates that PLXNB2 is expressed on keratinocytes and activatesSEMA4D-positive γδ T cells to contribute to epithelial repair (Witherdenet al., Immunity. 2012 Aug. 24; 37(2):314-25).

In lymphoid tissues, CD72 is utilized as a low affinity (300 nM) SEMA4Dreceptor (Kumanogoh et al., Immunity 13:621-631 (2000)). B cells andAntigen Presenting Cells (APC) express CD72, and anti-CD72 antibodieshave many of the same effects as sSEMA4D, such as enhancement ofCD40-induced B cell responses and B cell shedding of CD23. CD72 isthought to act as a negative regulator of B cell responses by recruitingthe tyrosine phosphatase SHP-1, which can associate with many inhibitoryreceptors. Interaction of SEMA4D with CD72 results in the dissociationof SHP-1, and the loss of this negative activation signal. SEMA4D hasbeen shown to promote T cell stimulation and B cell aggregation andsurvival in vitro. The addition of SEMA4D-expressing cells or sSEMA4Denhances CD40-induced B cell proliferation and immunoglobulin productionin vitro, and accelerates in vivo antibody responses (Ishida et al.,Inter. Immunol. 15:1027-1034 (2003); Kumanogoh and H. Kukutani, Trendsin Immunol. 22:670-676 (2001)). sSEMA4D enhances the CD40 inducedmaturation of DCs, including up-regulation of costimulatory moleculesand increased secretion of IL-12. In addition, sSEMA4D can inhibitimmune cell migration, which can be reversed by addition of blockinganti-SEMA4D mouse antibodies (Elhabazi et al., J. Immunol. 166:4341-4347(2001); Delaire et al., J. Immunol. 166:4348-4354 (2001)).

Sema4D is expressed at high levels in lymphoid organs, including thespleen, thymus, and lymph nodes, and in non-lymphoid organs, such as thebrain, heart, and kidney. In lymphoid organs, Sema4D is abundantlyexpressed on resting T cells but only weakly expressed on resting Bcells and antigen-presenting cells (APCs), such as dendritic cells(DCs).

Cellular activation increases the surface expression of SEMA4D as wellas the generation of soluble SEMA4D (sSEMA4D). The expression pattern ofSEMA4D suggests that it plays an important physiological as well aspathological role in the immune system. SEMA4D has been shown to promoteB cell activation, aggregation and survival; enhance CD40-inducedproliferation and antibody production; enhance antibody response to Tcell dependent antigens; increase T cell proliferation; enhancedendritic cell maturation and ability to stimulate T cells; and isdirectly implicated in demyelination and axonal degeneration (Shi etal., Immunity 13:633-642 (2000); Kumanogoh et al., J Immunol169:1175-1181 (2002); and Watanabe et al., J Immunol 167:4321-4328(2001)).

SEMA4D knock out (SEMA4D−/−) mice have provided additional evidence thatSEMA4D plays an important role in both humoral and cellular immuneresponses. There are no known abnormalities of non-lymphoid tissues inSEMA4D−/− mice. Dendritic cells (DCs) from the SEMA4D−/− mice have poorallostimulatory ability and show defects in expression of costimulatorymolecules, which can be rescued by the addition of sSEMA4D. Micedeficient in SEMA4D (SEMA4D−/−) fail to develop experimental autoimmuneencephalomyelitis induced by myelin oligodendrocyte glycoproteinpeptide, because myelin oligodendrocyte glycoprotein-specific T cellsare poorly generated in the absence of SEMA4D (Kumanogoh et al., JImmunol 169:1175-1181 (2002)). A significant amount of soluble SEMA4D isalso detected in the sera of autoimmunity-prone MRL/lpr mice (model ofsystemic autoimmune diseases such as SLE), but not in normal mice.Further, the levels of sSEMA4D correlate with levels of auto-antibodiesand increase with age (Wang et al., Blood 97:3498-3504 (2001)). SolubleSEMA4D has also been shown to accumulate in the cerebral spinal fluidand sera of patients with demyelinating disease, and sSEMA4D inducesapoptosis of human pluripotent neural precursors (Dev cells), and bothinhibits process extension and induces apoptosis of rat oligodendrocytesin vitro (Giraudon et al., J Immunol 172(2):1246-1255 (2004)). Thisapoptosis was blocked by an anti-SEMA4D monoclonal antibody (MAb).

Anti-SEMA4D Antibodies

Antibodies that bind SEMA4D have been described in the art. See, forexample, US Publ. Nos. 2008/0219971 A1, US 2010/0285036 A1, and US2006/0233793 A1, International Patent Applications WO 93/14125, WO2008/100995, and WO 2010/129917, and Herold et al., Int. Immunol. 7(1):1-8 (1995), each of which is herein incorporated in its entirety byreference.

The disclosure generally relates to a method of inhibiting, delaying, orreducing tumor growth or metastases in a subject, e.g., a human cancerpatient, comprising administration of an antibody which specificallybinds to SEMA4D, or an antigen-binding fragment, variant, or derivativethereof. In certain embodiments, the antibody blocks the interaction ofSEMA4D with one or more of its receptors, e.g., Plexin-B1 and/orPlexin-B2. In certain embodiments the cancer cells express Plexin-B1and/or Plexin-B2. Anti-SEMA4D antibodies having these properties can beused in the methods provided herein. Antibodies that can be usedinclude, but are not limited to MAbs VX15/2503, 67, 76, 2282 andantigen-binding fragments, variants, or derivatives thereof which arefully described in US 2010/0285036 A1 and US 2008/0219971 A1. Additionalantibodies which can be used in the methods provided herein include theBD16 antibody described in US 2006/0233793 A1 as well as antigen-bindingfragments, variants, or derivatives thereof; or any of MAb 301, MAb1893, MAb 657, MAb 1807, MAb 1656, MAb 1808, Mab 59, MAb 2191, MAb 2274,MAb 2275, MAb 2276, MAb 2277, MAb 2278, MAb 2279, MAb 2280, MAb 2281,MAb 2282, MAb 2283, MAb 2284, and MAb 2285, as well as any fragments,variants or derivatives thereof as described in US 2008/0219971 A1. Incertain embodiments an anti-SEMA4D antibody for use in the methodsprovided herein binds human, murine, or both human and murine SEMA4D.Also useful are antibodies which bind to the same epitope as any of theaforementioned antibodies and/or antibodies which competitively inhibitbinding or activity of any of the aforementioned antibodies.

In certain embodiments, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein has an amino acid sequence that has at least about 80%, about85%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, or about 95% sequence identity to the amino acid sequence fora reference anti-SEMA4D antibody molecule, for example, those describedabove. In a further embodiment, the binding molecule shares at leastabout 96%, about 97%, about 98%, about 99%, or 100% sequence identity toa reference antibody.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VH domain), where at leastone of the CDRs of the VH domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO:9, 10, 25, or 48.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VH domain), where at leastone of the CDRs of the VH domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VH domain), where at leastone of the CDRs of the VH domain has an amino acid sequence identical,except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, toSEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 27,or SEQ ID NO: 28.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of a VH domainthat has an amino acid sequence that is at least about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 25, or SEQ ID NO: 48, wherein an anti-SEMA4Dantibody comprising the encoded VH domain specifically or preferentiallybinds to SEMA4D.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VL domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO:17, 18, 29, or 47.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VL domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VL domain has an amino acid sequence identical,except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, toSEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 30, SEQ ID NO:31, or SEQ ID NO: 32.

In a further embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of a VL domainthat has an amino acid sequence that is at least about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 29, or SEQ ID NO: 47, wherein ananti-SEMA4D antibody comprising the encoded VL domain specifically orpreferentially binds to SEMA4D.

Also included for use in the methods provided herein are polypeptidesencoding anti-SEMA4D antibodies, or antigen-binding fragments, variants,or derivatives thereof as described herein, polynucleotides encodingsuch polypeptides, vectors comprising such polynucleotides, and hostcells comprising such vectors or polynucleotides, all for producinganti-SEMA4D antibodies, or antigen-binding fragments, variants, orderivatives thereof for use in the methods described herein.

Suitable biologically active variants of the anti-SEMA4D antibodies ofthe disclosure can be used in the methods of the present disclosure.Such variants will retain the desired binding properties of the parentanti-SEMA4D antibody. Methods for making antibody variants are generallyavailable in the art.

Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art. See, for example, Walker and Gaastra, eds. (1983)Techniques in Molecular Biology (MacMillan Publishing Company, NewYork); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel etal., Methods Enzymol. 154:367-382 (1987); Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S.Pat. No. 4,873,192; and the references cited therein; hereinincorporated by reference. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the polypeptideof interest can be found in the model of Dayhoff et al. (1978) in Atlasof Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), pp. 345-352, herein incorporated by reference in itsentirety. The model of Dayhoff et al. uses the Point Accepted Mutation(PAM) amino acid similarity matrix (PAM 250 matrix) to determinesuitable conservative amino acid substitutions. In certain aspects,conservative substitutions, such as exchanging one amino acid withanother having similar properties are used. Examples of conservativeamino acid substitutions as taught by the PAM 250 matrix of the Dayhoffet al. model include, but are not limited to, Gly4⇄Ala, Val⇄Ile⇄Leu,Asp⇄Glu, Lys⇄Arg, Asn⇄Gln, and Phe⇄Trp⇄Tyr.

In constructing variants of the anti-SEMA4D binding molecule, e.g., anantibody or antigen-binding fragment thereof, polypeptides of interest,modifications are made such that variants continue to possess thedesired properties, e.g., being capable of specifically binding to aSEMA4D, e.g., human, murine, or both human and murine SEMA4D, e.g.,expressed on the surface of or secreted by a cell and having SEMA4Dblocking activity, as described herein. In certain aspects, mutationsmade in the DNA encoding the variant polypeptide maintain the readingframe and do not create complementary regions that could producesecondary mRNA structure. See EP Patent Application Publication No.75,444.

Methods for measuring anti-SEMA4D binding molecule, e.g., an antibody orantigen-binding fragment, variant, or derivative thereof, bindingspecificity include, but are not limited to, standard competitivebinding assays, assays for monitoring immunoglobulin secretion by Tcells or B cells, T cell proliferation assays, apoptosis assays, ELISAassays, and the like. See, for example, such assays disclosed in WO93/14125; Shi et al., Immunity 13:633-642 (2000); Kumanogoh et al., JImmunol 169:1175-1181 (2002); Watanabe et al., J Immunol 167:4321-4328(2001); Wang et al., Blood 97:3498-3504 (2001); and Giraudon et al., JImmunol 172(2):1246-1255 (2004), all of which are herein incorporated byreference.

Methods for measuring the anti-angiogenic ability of an anti-SEMA4Dantibody or antigen-binding fragment, variant, or derivative thereof arewell known in the art.

When discussed herein whether any particular polypeptide, including theconstant regions, CDRs, VH domains, or VL domains disclosed herein, isat least about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or even about 100% identical to anotherpolypeptide, the % identity can be determined using methods and computerprograms/software known in the art such as, but not limited to, theBESTFIT program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). BESTFIT uses the local homology algorithmof Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, to find thebest segment of homology between two sequences. When using BESTFIT orany other sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present disclosure, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

For purposes of the present disclosure, percent sequence identity can bedetermined using the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology searchalgorithm is taught in Smith and Waterman (1981) Adv. Appl. Math.2:482-489. A variant can, for example, differ from a referenceanti-SEMA4D antibody (e.g., MAb VX15/2503, 67, 76, or 2282) by as few as1 to 15 amino acid residues, as few as 1 to 10 amino acid residues, suchas 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The constant region of an anti-SEMA4D antibody can be mutated to altereffector function in a number of ways. For example, see U.S. Pat. No.6,737,056B1 and U.S. Patent Application Publication No. 2004/0132101A1,which disclose Fc mutations that optimize antibody binding to Fcreceptors.

In certain anti-SEMA4D antibodies or fragments, variants or derivativesthereof useful in the methods provided herein, the Fc portion can bemutated to decrease effector function using techniques known in the art.For example, the deletion or inactivation (through point mutations orother means) of a constant region domain can reduce Fc receptor bindingof the circulating modified antibody thereby increasing tumorlocalization. In other cases, constant region modifications consistentwith the instant disclosure moderate complement binding and thus reducethe serum half-life. Yet other modifications of the constant region canbe used to modify disulfide linkages or oligosaccharide moieties thatallow for enhanced localization due to increased antigen specificity orantibody flexibility. The resulting physiological profile,bioavailability and other biochemical effects of the modifications, suchas tumor localization, biodistribution and serum half-life, can easilybe measured and quantified using well known immunological techniqueswithout undue experimentation.

Anti-SEMA4D antibodies for use in the methods provided herein includederivatives that are modified, e.g., by the covalent attachment of anytype of molecule to the antibody such that covalent attachment does notprevent the antibody from specifically binding to its cognate epitope.For example, but not by way of limitation, the antibody derivativesinclude antibodies that have been modified, e.g., by glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. Any of numerous chemicalmodifications can be carried out by known techniques, including, but notlimited to specific chemical cleavage, acetylation, formylation, etc.Additionally, the derivative can contain one or more non-classical aminoacids.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind an anti-SEMA4D polypeptide, to block SEMA4D interactionwith its receptor, or to inhibit, delay, or reduce metastases in asubject, e.g., a cancer patient).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations can be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations can be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations can alter an antibody's ability to bind antigen. One of skillin the art would be able to design and test mutant molecules withdesired properties such as no alteration in antigen binding activity oralteration in binding activity (e.g., improvements in antigen bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein can routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of a SEMA4D polypeptide)can be determined using techniques described herein or by routinelymodifying techniques known in the art.

In certain embodiments, the anti-SEMA4D antibodies for use in themethods provided herein comprise at least one optimizedcomplementarity-determining region (CDR). By “optimized CDR” is intendedthat the CDR has been modified and optimized to improve binding affinityand/or anti-SEMA4D activity that is imparted to an anti-SEMA4D antibodycomprising the optimized CDR. “Anti-SEMA4D activity” or “SEMA4D blockingactivity” can include activity which modulates one or more of thefollowing activities associated with SEMA4D: B cell activation,aggregation and survival; CD40-induced proliferation and antibodyproduction; antibody response to T cell dependent antigens; T cell orother immune cell proliferation; dendritic cell maturation;demyelination and axonal degeneration; apoptosis of pluripotent neuralprecursors and/or oligodendrocytes; induction of endothelial cellmigration; inhibition of spontaneous monocyte migration; inhibition,delay, or reduction of tumor cell growth or metastasis, binding to cellsurface Plexin B1 or other receptor, or any other activity associationwith soluble SEMA4D or SEMA4D that is expressed on the surface ofSEMA4D+ cells. In a particular embodiment, anti-SEMA4D activity includesthe ability to inhibit, delay, or reduce tumor metastases, either incombination with inhibition, delay, or reduction of primary tumor cellgrowth and tumor metastases, or independently of primary tumor cellgrowth and tumor metastases. Anti-SEMA4D activity can also be attributedto a decrease in incidence or severity of diseases associated withSEMA4D expression, including, but not limited to, certain types ofcancers including lymphomas, autoimmune diseases, inflammatory diseasesincluding central nervous system (CNS) and peripheral nervous system(PNS) inflammatory diseases, transplant rejections, and invasiveangiogenesis. Examples of optimized antibodies based on murineanti-SEMA4D MAb BD16 were described in US Publ. No. 2008/0219971 A1,International Patent Application WO 93/14125 and Herold et al., Int.Immunol. 7(1): 1-8 (1995), each of which are herein incorporated byreference in their entirety. The modifications can involve replacementof amino acid residues within the CDR such that an anti-SEMA4D antibodyretains specificity for the SEMA4D antigen and has improved bindingaffinity and/or improved anti-SEMA4D activity.

Binding Characteristics of Anti-SEMA4D Antibodies

In certain embodiments the binding molecule is an antibody whichspecifically binds to SEMA4D, or an antigen-binding fragment, variant,or derivative thereof. In certain embodiments, the binding moleculebinds to an epitope of SEMA4D. The nucleotide and amino acid sequencesfor one variant of SEMA4D are set forth in SEQ ID NO: 13 and SEQ ID NO:14, respectively, and for another variant of SEMA4D are set forth in SEQID NO: 15 and SEQ ID NO: 16. In some embodiments, the anti-SEMA4Dantibody designated as VX15/2503 is provided. Antibodies that have thebinding characteristics of antibody VX15/2503 are also disclosed herein.Such antibodies include, but are not limited to, antibodies that competein competitive binding assays with VX15/2503, as well as antibodies thatbind to an epitope (as defined below) capable of binding VX15/2503.Methods for assessing whether antibodies have the same or similarbinding characteristics include traditional quantitative methods suchas, for example, determining and comparing antibody affinity or avidityfor the antigenic epitope (e.g., SEMA4D peptide). Other exemplarymethods for comparing the binding characteristics of antibodies includecompetitive western blotting, enzyme immunoassays, ELISA, and flowcytometry. Methods for assessing and comparing antibody-antigen bindingcharacteristics are well known in the art. Variants and fragments ofVX15/2503 that retain the ability to specifically bind to SEMA4D arealso provided. Antibodies VX15/2503 and 67 share the same 6 CDRs andbind the same SEMA4D epitope.

In some embodiments, anti-SEMA4D antibodies, or antigen-bindingfragments, variants, or derivatives thereof disclosed herein can bedescribed or specified in terms of the epitope(s) or portion(s) of anantigen, e.g., a target polypeptide disclosed herein (e.g., SEMA4D) thatthey recognize or specifically bind. The portion of a target polypeptidethat specifically interacts with the antigen binding domain of anantibody is an “epitope,” or an “antigenic determinant.”

In some embodiments, an “epitope” is intended to be the part of anantigenic molecule which is used to produce an antibody and/or to whichan antibody will specifically bind. A “SEMA4D epitope” comprises thepart of the SEMA4D protein to which an anti-SEMA4D antibody binds.Epitopes can comprise linear amino acid residues (i.e., residues withinthe epitope that are arranged sequentially one after another in a linearfashion), nonlinear amino acid residues (referred to herein as“nonlinear epitopes” or “conformational epitopes”; these epitopes arenot arranged sequentially), or both linear and nonlinear amino acidresidues. Nonlinear epitopes or conformational epitopes can also includeamino acid residues that contribute to the overall conformation of therecognition structure of the antibody, but do not necessarily bind theantibody. Typically, epitopes are short amino acid sequences, e.g.,about five amino acids in length. Systematic techniques for identifyingepitopes are known in the art and are described, for example, in theexamples set forth below.

A target polypeptide can comprise a single epitope, but typicallycomprises at least two epitopes, and can include any number of epitopes,depending on the size, conformation, and type of antigen. Furthermore,it should be noted that an “epitope” on a target polypeptide can be orcan include non-polypeptide elements, e.g., an epitope can include acarbohydrate side chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes can contain at least seven, at least nine, or at least about 15to about 30 amino acids. Since a CDR can recognize an antigenic peptideor polypeptide in its tertiary form, the amino acids comprising anepitope need not be contiguous, and in some cases, may not even be onthe same peptide chain. A peptide or polypeptide epitope recognized byanti-SEMA4D antibodies of the present disclosure can contain a sequenceof at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 15, at least 20, at least 25, or between about15 to about 30 contiguous or non-contiguous amino acids of SEMA4D.

In some embodiments, the epitope has at least 80%, 85%, 90%, 95%, or100% identity to a target polypeptide amino acid sequence (e.g., thesequence set forth in SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46).

In some embodiments, the epitope is identical to a target polypeptideamino acid sequence (e.g., the sequence set forth in SEQ ID NO: 42, SEQID NO: 44, or SEQ ID NO: 46) except for 4, 3, 2, 1 or 0 amino acidsubstitutions. In another embodiment, the epitope is identical to atarget polypeptide amino acid sequence (e.g., the sequence set forth inSEQ ID NO: 42, SEQ ID NO: 44, or SEQ ID NO: 46) except for conservativeamino acid substitutions (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0conservative amino acid substitutions).

In some embodiments, the epitope comprises a sequence set forth in SEQID NO: 42, SEQ ID NO: 44, or SEQ ID NO: 46. In another embodiment, theepitope is the sequence set forth in SEQ ID NO: 42, SEQ ID NO: 44, orSEQ ID NO: 46. In some embodiments, the epitope is a linear epitope. Insome embodiments, the epitope is a conformational epitope.

In some embodiments, the epitope comprises, consists essentially of, orconsists of LKVPVFYALFTPQLNNV (SEQ ID NO: 42, corresponding to residues304 through 320 of the full-length SEMA4D amino acid sequence set forthin SEQ ID NO: 1), KWTSFLKARLIASRP (SEQ ID NO: 44, corresponding toresidues 270 through 284 of the full-length SEMA4D amino acid sequenceset forth in SEQ ID NO: 1, wherein position 281 can be a cysteine or analanine), or EFVFRVLIPRIARV (SEQ ID NO: 46; corresponding to residues243 through 256 of the full-length SEMA4D amino acid sequence set forthin SEQ ID NO: 1). In some embodiments, the epitope comprises one or moreof the amino acid sequences set forth in SEQ ID NO: 42, 44 and 46. Insome embodiments, the epitope is a discontinuous epitope comprised inthe domain spanning amino acid residues 243 to 320 of SEQ ID NO: 1.

Treatment Methods Using Therapeutic Anti-SEMA4D Antibodies as a SingleAgent or in Combination with at Least One Immune Modulating Therapy

Methods of the disclosure are directed to the use of anti-SEMA4D oranti-Plexin-B1 binding molecules, e.g., antibodies, includingantigen-binding fragments, variants, and derivatives thereof, either assingle agents or in combination with at least one other immunemodulating therapy, to inhibit, delay, or reduce tumor growth ormetastases in a subject in need of such inhibition, delay, or reduction,e.g., a cancer patient. In certain embodiments the cancer cells expressa SEMA4D receptor, in certain embodiments the receptor is Plexin-B1.Though the following discussion refers to administration of ananti-SEMA4D antibody, the methods described herein are equallyapplicable to the antigen-binding fragments, variants, and derivativesof these antibodies that retain the desired properties of the antibodiesof the disclosure, e.g., capable of specifically binding SEMA4D, e.g.,human, mouse, or human and mouse SEMA4D, having SEMA4D neutralizingactivity, and/or blocking the interaction of SEMA4D with its receptors.The methods described herein are also applicable to other biologicproducts or small molecule drugs that retain the desired properties ofthe antibodies of the disclosure, e.g., capable of specifically bindingSEMA4D, e.g., human, mouse, or human and mouse SEMA4D, having SEMA4Dneutralizing activity, and/or blocking the interaction of SEMA4D withits receptors.

In one embodiment, anti-SEMA4D molecules, e.g., antibodies, includingantigen-binding fragments, variants, and derivatives thereof, can beused as a single agent to inhibit, delay, or reduce tumor growth in asubject in need of such inhibition, delay, or reduction, e.g., a cancerpatient. In certain embodiments, the cancer cells express a SEMA4Dreceptor, such as, for example, Plexin-B1 or Plexin-B2. In otherembodiments, the cancer cells express other receptors that can work inconjunction with a SEMA4D receptor. An example of such a receptor isHER2 (ErbB2). Examples of cancers in which expression of Plexin-B1 orPlexin-B2 in combination with Her2 has been observed include lungcancer, breast cancer, prostate cancer, and ovarian cancer. As such, incertain embodiments anti-SEMA4D molecules, e.g., antibodies, includingantigen-binding fragments, variants, and derivatives thereof, can beused as a single agent to inhibit, delay, or reduce tumor growth in asubject having lung cancer, breast cancer, prostate cancer, or ovariancancer.

In one embodiment, the immune modulating therapy can include cancervaccines, immunostimulatory agents, adoptive T cell or antibody therapy,and inhibitors of immune checkpoint blockade (Lizée et al. 2013.Harnessing the Power of the Immune System to Target Cancer. Annu. Rev.Med. Vol. 64 No. 71-90).

Cancer Vaccines. Cancer vaccines activate the body's immune system andnatural resistance to an abnormal cell, such as cancer, resulting ineradication or control of the disease. Cancer vaccines generally consistof a tumor antigen in an immunogenic formulation that activates tumorantigen-specific helper cells and/or CTLs and B cells. Vaccines can bein a variety of formulations, including, but not limited to, dendriticcells, especially autologous dendritic cells pulsed with tumor cells ortumor antigens, heterologous tumor cells transfected with an immunestimulating agent such as GM-CSF, recombinant virus, or proteins orpeptides that are usually administered together with a potent immuneadjuvant such as CpG.

Immunostimulatory Agents. Immunostimulatory agents act to enhance orincrease the immune response to tumors, which is suppressed in manycancer patients through various mechanisms. Immune modulating therapiescan target lymphocytes, macrophages, dendritic cells, natural killercells (NK Cell), or subsets of these cells such as cytotoxic Tlymphocytes (CTL) or Natural Killer T (NKT) cells. Because ofinteracting immune cascades, an effect on one set of immune cells willoften be amplified by spreading to other cells, e.g. enhanced antigenpresenting cell activity promotes response of T and B lymphocytes.Examples of immunostimulatory agents include, but are not limited to,HER2, cytokines such as G-CSF, GM-CSF and IL-2, cell membrane fractionsfrom bacteria, glycolipids that associate with CD1d to activate NaturalKiller T (NKT) cells, CpG oligonucleotides.

Macrophages, myelophagocytic cells of the immune system, are afundamental part of the innate defense mechanisms, which can promotespecific immunity by inducing T cell recruitment and activation. Despitethis, their presence within the tumor microenvironment has beenassociated with enhanced tumor progression and shown to promote cancercell growth and spread, angiogenesis and immunosuppression. Key playersin the setting of their phenotype are the microenvironmental signals towhich macrophages are exposed, which selectively tune their functionswithin a functional spectrum encompassing the M1 (tumor inhibitingmacrophage) and M2 (tumor promoting macrophage) extremes. Sica et al.,Seminars in Cancer Biol. 18:349-355 (2008). Increased macrophage numbersduring cancer generally correlates with poor prognosis (Qualls andMurray, Curr. Topics in Develop. Biol. 94:309-328 (2011)). Of themultiple unique stromal cell types common to solid tumors,tumor-associated macrophages (TAMs) are significant for fostering tumorprogression. Targeting molecular pathways regulating TAM polarizationholds great promise for anticancer therapy. Ruffell et al., Trends inImmunol. 33:119-126 (2012).

Adoptive Cell Transfer. Adoptive cell transfer can employ T cell-basedcytotoxic responses to attack cancer cells. Autologous T cells that havea natural or genetically engineered reactivity to a patient's cancer aregenerated and expanded in vitro and then transferred back into thecancer patient. One study demonstrated that adoptive transfer of invitro expanded autologous tumor-infiltrating lymphocytes was aneffective treatment for patients with metastatic melanoma. (Rosenberg SA, Restifo N P, Yang J C, Morgan R A, Dudley M E (April 2008). “Adoptivecell transfer: a clinical path to effective cancer immunotherapy”. Nat.Rev. Cancer 8 (4): 299-308). This can be achieved by taking T cells thatare found within resected patient tumor. These T cells are referred toas tumor-infiltrating lymphocytes (TIL) and are presumed to havetrafficked to the tumor because of their specificity for tumor antigens.Such T cells can be induced to multiply in vitro using highconcentrations of IL-2, anti-CD3 and allo-reactive feeder cells. These Tcells are then transferred back into the patient along with exogenousadministration of IL-2 to further boost their anti-cancer activity. Inother studies, autologous T cells have been transduced with a chimericantigen receptor that renders them reactive to a targeted tumor antigen(Liddy et al., Nature Med. 18:980-7, (2012); Grupp et al., New EnglandJ. Med. 368:1509-18, (2013)).

Other adoptive cell transfer therapies employ autologous dendritic cellsexposed to natural or modified tumor antigens ex vivo that arere-infused into the patient. Provenge is such an FDA approved therapy inwhich autologous cells are incubated with a fusion protein of prostaticacid phosphatase and GM-CSF to treat patients with prostate tumors.GM-CSF is thought to promote the differentiation and activity of antigenpresenting dendritic cells (Small et al., J. Clin. Oncol. 18:3894-903(2000); U.S. Pat. No. 7,414,108)).

Immune Checkpoint Blockade Immune checkpoint blockade therapies enhanceT-cell immunity by removing a negative feedback control that limitsongoing immune responses. These types of therapies target inhibitorypathways in the immune system that are crucial for modulating theduration and amplitude of physiological immune responses in peripheraltissues (anti-CTLA4) or in tumor tissue expressing PD-L1 (anti-PD1 oranti-PD-L1) in order to minimize collateral tissue damage. Tumors canevolve to exploit certain immune-checkpoint pathways as a majormechanism of immune resistance against T cells that are specific fortumor antigens. Since many immune checkpoints are initiated byligand-receptor interactions, these checkpoints can be blocked byantibodies to either receptor or ligand or can be modulated by solublerecombinant forms of the ligands or receptors. Neutralization of immunecheckpoints allows tumor-specific T cells to continue to function in theotherwise immunosuppressive tumor microenvironment. Examples of immunecheckpoint blockade therapies are those which target CytotoxicT-lymphocyte-associated antigen 4 (CTLA-4), PD-1, its ligand PD-L1, LAG3and B7-H3.

Cyclophosphamide. Cyclophosphamide, a commonly used chemotherapeuticagent, can enhance immune responses. Cyclophosphamide differentiallysuppresses the function of regulatory T cells (Tregs) relative toeffector T cells. Tregs are important in regulating anticancer immuneresponses. Tumor-infiltrating Tregs have previously been associated withpoor prognosis. While agents that target Tregs specifically arecurrently unavailable, cyclophosphamide has emerged as a clinicallyfeasible agent that can preferentially suppress Tregs relative to otherT cells and, therefore, allows more effective induction of antitumorimmune responses.

Other Immune-Modulating Therapies: In another embodiment, therapy with aSEMA4D or Plexin-B1 binding molecule, e.g., an antibody or antigenbinding fragment, variant, or derivative thereof, can be combined witheither low dose chemotherapy or radiation therapy. Although standardchemotherapy is often immunosuppressive, low doses of chemotherapeuticagents such as cyclophosphamide, doxorubicin, and paclitaxel have beenshown to enhance responses to vaccine therapy for cancer (Machiels etal., Cancer Res. 61:3689-3697 (2001)). In some cases, chemotherapy candifferentially inactivate T regulatory cells (Treg) and myeloid derivedsuppressor cells (MDSC) that negatively regulate immune responses in thetumor environment. Radiation therapy has been generally employed toexploit the direct tumorcidal effect of ionizing radiation. Indeed, highdose radiation can, like chemotherapy, be immunosuppressive. Numerousobservations, however, suggest that under appropriate conditions of dosefractionation and sequencing, radiation therapy can enhancetumor-specific immune responses and the effects of immune modulatingagents. One of several mechanisms that contribute to this effect iscross-presentation by dendritic cells and other antigen presenting cellsof tumor antigens released by radiation-induced tumor-cell death(Higgins et al., Cancer Biol. Ther. 8:1440-1449 (2009)). In effect,radiation therapy can induce in situ vaccination against a tumor (Ma etal., Seminar Immunol. 22:113-124 (2010)) and this could be amplified bycombination with therapy with a SEMA4D or Plexin-B1 binding molecule,e.g., an antibody or antigen binding fragment, variant, or derivativethereof.

In one embodiment, the immune modulating therapy can be an immunemodulating agent, including, but not limited to, interleukins such asIL-2, IL-7, IL-12; cytokines such as granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferons; various chemokines suchas CXCL13, CCL26, CXCL7; antagonists of immune checkpoint blockades suchas anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-LAG3 and anti-B7-H3;synthetic cytosine phosphate-guanosine (CpG), oligodeoxynucleotides,glucans, modulators of regulatory T cells (Tregs) such ascyclophosphamide, or other immune modulating agents. In one embodiment,the immune modulating agent is an agonist antibody to 4-1BB (CD137). Asrecently reported, such agonist antibody to 4-1BB can give rise to anovel class of KLRG1+ T cells that are highly cytotoxic for tumors(Curran et al., J. Exp. Med. 210:743-755 (2013)). In all cases, theadditional immune modulating therapy is administered prior to, during,or subsequent to the anti-SEMA4D or anti-Plexin-B1 binding molecule,e.g., antibody or antigen binding fragment, variant, or derivativethereof, therapy. Where the combined therapies comprise administrationof an anti-SEMA4D binding molecule, e.g., an antibody or antigen bindingfragment, variant, or derivative thereof, in combination withadministration of another immune modulating agent, the methods of thedisclosure encompass co-administration, using separate formulations or asingle pharmaceutical formulation, with simultaneous or consecutiveadministration in either order.

In one embodiment, the immune modulating therapy can be a cancer therapyagent, including, but not limited to, surgery or surgical procedures(e.g. splenectomy, hepatectomy, lymphadenectomy, leukophoresis, bonemarrow transplantation, and the like); radiation therapy; chemotherapy,optionally in combination with autologous bone marrow transplant, orother cancer therapy; where the additional cancer therapy isadministered prior to, during, or subsequent to the anti-SEMA4D bindingmolecule, e.g., antibody or antigen binding fragment, variant, orderivative thereof, therapy. Where the combined therapies compriseadministration of an anti-SEMA4D binding molecule, e.g., an antibody orantigen binding fragment, variant, or derivative thereof, in combinationwith administration of another therapeutic agent, the methods of thedisclosure encompass co-administration, using separate formulations or asingle pharmaceutical formulation, with simultaneous or consecutiveadministration in either order.

In another embodiment, the disclosure is directed to the use ofanti-SEMA4D or anti-Plexin-B1 binding molecules, e.g., antibodies,including antigen-binding fragments, variants, and derivatives thereof,either as single agents or in combination with at least one other immunemodulating therapy, to treat cancer patients with elevated levels ofeither B cells, T cells or both B cells and T cells in circulation whencompared to other patients with solid tumors, such as those found in thebrain, ovary, breast, colon and other tissues but excludinghematological cancers. As used herein, the term “elevated” refers tocancer patients that have at least 1.5 times, e.g., about 1.5 to about 5times, e.g., about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 or more times themean number of B cells and/or T cells in circulation than other cancerpatients. In one non-limiting example, in a group of 34 patients withsolid tumors, the mean number of B cells was 98 per microliter of bloodand the mean number of T cells was 782 per microliter of blood.Accordingly, the mean number of B cells and T cells per microliter ofblood observed in this subset of cancer patients with elevated B celland T cell levels can range from about 147 to about 588 and from about1173 to about 3910, respectively, when compared to other cancerpatients.

In another embodiment, the disclosure is directed to the use ofanti-SEMA4D or anti-Plexin-B1 binding molecules, e.g., antibodies,including antigen-binding fragments, variants, and derivatives thereof,either as single agents or in combination with at least one other immunemodulating therapy, to treat cancer patients with levels of either Bcells, T cells or both B cells and T cells in circulation that fallwithin or above the range of normal individuals. As used herein, theterm “normal” refers to the B and/or T cell levels that are found inhealthy, non-cancer patients. As used herein, the term “within” refersto a ten (10) percent difference in B and/or T cell levels. In onenon-limiting example, the range of normal levels include, for instance,a B cell count of about 250 cells per microliter or more and/or a T cellcount of about 1500 cells per microliter or more. Therefore, the meannumber of B cells and T cells per microliter of blood in cancer patientswith elevated B cell and T cell levels can range from about 225 to about275 or more and from about 1350 to about 1650 and more, respectively,when compared to healthy, non-cancer patients. Of course, one skilled inthe art should appreciate that the levels of B and T cells can varydepending on a variety of factors, e.g., type of cancer, stage ofcancer, etc., and, therefore, levels that are below the ones providedabove can also constitute elevated levels for a certain type or stage ofcancer.

In some embodiments, the absolute T and B cell counts are measured usinga validated flow cytometric-based immunophenotypic assay (BD Mutitest6-color TBNK Reagent), which is a six color direct immunofluorescentassay that also utilizes BD Trucount tubes and a BD FACScanto flowcytometer. This assay is used routinely to determine the percentages andabsolute counts of T, B, and NK cells as well as CD4 and CD8subpopulations of T cells in peripheral blood. Peripheral blood cellsare first gated on CD45+ lymphocytes. T cells are defined as CD3+ cellswithin this gate and B cells are defined as CD19+ CD3− cells within thisgate. Percentages are simply taken directly from the flow cytometerafter the appropriate gate is set, and the absolute counts arecalculated using the following formula (taken directly from the BDprocedure manual): [(#events in cell population/#events in absolutecount bead region)]*[(#beads/testa)/test volume]=cell populationabsolute count, where “a” is the value found on the BD Trucount tubefoil pouch label.

It should also be appreciated that the methods described herein are alsoapplicable to the substitution of anti-Plexin-B1 binding molecules foranti-SEMA4D binding molecules. In some embodiments, an anti-Plexin-B1binding molecule can be used to inhibit the interaction of SEMA4D withPlexin-B1 by blocking binding of SEMA4D to Plexin-B1 and/or bypreventing activation of Plexin-B1 by SEMA4D. It should also beappreciated that the methods described herein are also applicable to theuse of small molecule drugs or other biologic products to inhibit theactivity of SEMA4D or Plexin-B1. In some embodiments, a small moleculedrug or a biologic product other than an anti-SEMA4D binding moleculecan be used to inhibit the interaction of SEMA4D with Plexin-B1 byblocking binding of SEMA4D to Plexin-B1 and/or by preventing activationof Plexin-B1 by SEMA4D.

In one embodiment, treatment includes the application or administrationof an anti-SEMA4D binding molecule, e.g., an antibody or antigen bindingfragment thereof as described herein as a single agent or in combinationwith at least one other immune modulating therapy to a patient, orapplication or administration of the anti-SEMA4D binding molecule as asingle agent or in combination with at least one other immune modulatingtherapy to an isolated tissue or cell line from a patient, where thepatient has, or has the risk of developing metastases of cancer cells.In another embodiment, treatment is also intended to include theapplication or administration of a pharmaceutical composition comprisingthe anti-SEMA4D binding molecules, e.g., an antibody or antigen bindingfragment thereof to a patient, in combination with at least one otherimmune modulating therapy or application or administration of apharmaceutical composition comprising the anti-SEMA4D binding moleculeand at least one other immune modulating therapy to an isolated tissueor cell line from a patient, where the patient has, or has the risk ofdeveloping metastases of cancer cells.

The anti-SEMA4D binding molecules, e.g., antibodies or binding fragmentsthereof as described herein, as single agents or in combination with atleast one other immune modulating therapy are useful for the treatmentof various malignant and non-malignant tumors. By “anti-tumor activity”is intended a reduction in the rate of SEMA4D production or accumulationassociated directly with the tumor or indirectly with stromal cells ofthe tumor environment, and hence a decline in growth rate of an existingtumor or of a tumor that arises during therapy, and/or destruction ofexisting neoplastic (tumor) cells or newly formed neoplastic cells, andhence a decrease in the overall size of a tumor and/or the number ofmetastatic sites during therapy. For example, therapy with at least oneanti-SEMA4D antibody as a single agent or in combination with at leastone other immune modulating therapy causes a physiological response, forexample, a reduction in metastases, that is beneficial with respect totreatment of disease states associated with SEMA4D -expressing cells ina human.

In one embodiment, the disclosure relates to the use of anti-SEMA4Dbinding molecules, e.g., antibodies or antigen-binding fragments,variants, or derivatives thereof, as a single agent or in combinationwith at least one other immune modulating therapy as a medicament, inthe treatment or prophylaxis of cancer or for use in a precancerouscondition or lesion to inhibit, reduce, prevent, delay, or minimalizethe growth or metastases of tumor cells.

In accordance with the methods of the present disclosure, at least oneanti-SEMA4D binding molecule, e.g., an antibody or antigen bindingfragment, variant, or derivative thereof, as a single agent or incombination with at least one other immune modulating therapy can beused to promote a positive therapeutic response with respect to amalignant human cell. By “positive therapeutic response” with respect tocancer treatment is intended an improvement in the disease inassociation with the anti-tumor activity of these binding molecules,e.g., antibodies or fragments thereof, and/or an improvement in thesymptoms associated with the disease. In particular, the methodsprovided herein are directed to inhibiting, preventing, reducing,alleviating, delaying, or lessening growth of a tumor and/or thedevelopment of metastases of primary tumors in a patient. That is theprevention of distal tumor outgrowths, can be observed. Thus, forexample, an improvement in the disease can be characterized as acomplete response. By “complete response” is intended an absence ofclinically detectable metastases with normalization of any previouslyabnormal radiographic studies, e.g. at the site of the primary tumor orthe presence of tumor metastases in bone marrow. Alternatively, animprovement in the disease can be categorized as being a partialresponse. By “partial response” is intended at least about a 50%decrease in all measurable metastases (i.e., the number of tumor cellspresent in the subject at a remote site from the primary tumor).Alternatively, an improvement in the disease can be categorized as beingrelapse free survival or “progression free survival”. By “relapse freesurvival” is intended the time to recurrence of a tumor at any site.“Progression free survival” is the time before further growth of tumorat a site being monitored can be detected.

Inhibition, delay, or reduction of metastases can be assessed usingscreening techniques such as imaging, for example, fluorescent antibodyimaging, bone scan imaging, and tumor biopsy sampling including bonemarrow aspiration (BMA), or immunohistochemistry. In addition to thesepositive therapeutic responses, the subject undergoing therapy with theanti-SEMA4D binding molecule, e.g., an antibody or antigen-bindingfragment, variant, or derivative thereof, can experience the beneficialeffect of an improvement in the symptoms associated with the disease.

Clinical response can be assessed using screening techniques such asmagnetic resonance imaging (MRI) scan, x-radiographic imaging, computedtomographic (CT) scan, flow cytometry or fluorescence-activated cellsorter (FACS) analysis, histology, gross pathology, and blood chemistry,including but not limited to changes detectable by ELISA, RIA,chromatography, and the like.

To apply the methods and systems of the disclosure in certainembodiments, samples from a patient can be obtained before or after theadministration of a therapy comprising either: (1) an effective amountof an isolated binding molecule that specifically binds to semaphorin-4D(SEMA4D) and an effective amount of at least one other immune modulatingtherapy; or (2) an effective amount of an isolated binding molecule thatspecifically binds to semaphorin-4D (SEMA4D) to a subject having a tumorthat is Her2+ and either Plexin B1+ or Plexin B2+. In some cases,successive samples can be obtained from the patient after therapy hascommenced or after therapy has ceased. Samples can, for example, berequested by a healthcare provider (e.g., a doctor) or healthcarebenefits provider, obtained and/or processed by the same or a differenthealthcare provider (e.g., a nurse, a hospital) or a clinicallaboratory, and after processing, the results can be forwarded to yetanother healthcare provider, healthcare benefits provider or thepatient. Similarly, the measuring/determination of one or more scores,comparisons between scores, evaluation of the scores and treatmentdecisions can be performed by one or more healthcare providers,healthcare benefits providers, and/or clinical laboratories.

As used herein, the term “healthcare provider” refers to individuals orinstitutions that directly interact and administer to living subjects,e.g., human patients. Non-limiting examples of healthcare providersinclude doctors, nurses, technicians, therapist, pharmacists,counselors, alternative medicine practitioners, medical facilities,doctor's offices, hospitals, emergency rooms, clinics, urgent carecenters, alternative medicine clinics/facilities, and any other entityproviding general and/or specialized treatment, assessment, maintenance,therapy, medication, and/or advice relating to all, or any portion of, apatient's state of health, including but not limited to general medical,specialized medical, surgical, and/or any other type of treatment,assessment, maintenance, therapy, medication and/or advice.

In some aspects, a healthcare provider can administer or instructanother healthcare provider to administer a therapy comprising either:(1) an effective amount of an isolated binding molecule thatspecifically binds to semaphorin-4D (SEMA4D) and an effective amount ofat least one other immune modulating therapy; or (2) an effective amountof an isolated binding molecule that specifically binds to semaphorin-4D(SEMA4D), where the subject has, or is suspected to have, tumor cellsthat are Her2+ and either Plexin B1+ or Plexin B2+. A healthcareprovider can implement or instruct another healthcare provider orpatient to perform the following actions: obtain a sample, process asample, submit a sample, receive a sample, transfer a sample, analyze ormeasure a sample, quantify a sample, provide the results obtained afteranalyzing/measuring/quantifying a sample, receive the results obtainedafter analyzing/measuring/quantifying a sample, compare/score theresults obtained after analyzing/measuring/quantifying one or moresamples, provide the comparison/score from one or more samples, obtainthe comparison/score from one or more samples, administer a therapy(e.g., (1) an effective amount of an isolated binding molecule thatspecifically binds to semaphorin-4D (SEMA4D) and an effective amount ofat least one other immune modulating therapy; or (2) an effective amountof an isolated binding molecule that specifically binds to semaphorin-4D(SEMA4D) to a subject, where the subject has, or is suspected to have,tumor cells that are Her2+ and either Plexin B1+ or Plexin B2+, commencethe administration of a therapy, cease the administration of a therapy,continue the administration of a therapy, temporarily interrupt theadministration of a therapy, increase the amount of an administeredtherapeutic agent, decrease the amount of an administered therapeuticagent, continue the administration of an amount of a therapeutic agent,increase the frequency of administration of a therapeutic agent,decrease the frequency of administration of a therapeutic agent,maintain the same dosing frequency on a therapeutic agent, replace atherapy or therapeutic agent by at least another therapy or therapeuticagent, combine a therapy or therapeutic agent with at least anothertherapy or additional therapeutic agent. In some aspects, a healthcarebenefits provider can authorize or deny, for example, collection of asample, processing of a sample, submission of a sample, receipt of asample, transfer of a sample, analysis or measurement a sample,quantification a sample, provision of results obtained afteranalyzing/measuring/quantifying a sample, transfer of results obtainedafter analyzing/measuring/quantifying a sample, comparison/scoring ofresults obtained after analyzing/measuring/quantifying one or moresamples, transfer of the comparison/score from one or more samples,administration of a therapy or therapeutic agent, commencement of theadministration of a therapy or therapeutic agent, cessation of theadministration of a therapy or therapeutic agent, continuation of theadministration of a therapy or therapeutic agent, temporary interruptionof the administration of a therapy or therapeutic agent, increase of theamount of administered therapeutic agent, decrease of the amount ofadministered therapeutic agent, continuation of the administration of anamount of a therapeutic agent, increase in the frequency ofadministration of a therapeutic agent, decrease in the frequency ofadministration of a therapeutic agent, maintain the same dosingfrequency on a therapeutic agent, replace a therapy or therapeutic agentby at least another therapy or therapeutic agent, or combine a therapyor therapeutic agent with at least another therapy or additionaltherapeutic agent.

In addition a healthcare benefits provides can, e.g., authorize or denythe prescription of a therapy, authorize or deny coverage for therapy,authorize or deny reimbursement for the cost of therapy, determine ordeny eligibility for therapy, etc.

In some aspects, a clinical laboratory can, for example, collect orobtain a sample, process a sample, submit a sample, receive a sample,transfer a sample, analyze or measure a sample, quantify a sample,provide the results obtained after analyzing/measuring/quantifying asample, receive the results obtained afteranalyzing/measuring/quantifying a sample, compare/score the resultsobtained after analyzing/measuring/quantifying one or more samples,provide the comparison/score from one or more samples, obtain thecomparison/score from one or more samples, or other related activities.

Methods of Diagnosis and Treatment

In certain embodiments, this disclosure provides methods of treating asubject, e.g., a cancer patient, where the subject has elevated levelsof either B cells, T cells or both B cells and T cells, comprisingadministering a combination of an effective amount of an isolatedbinding molecule that specifically binds to semaphorin-4D (SEMA4D) andan effective amount of at least one other immune modulating therapy ifthe subject's B cell, T cell or both B cell and T cell levels are abovea predetermined threshold level of B cells, T cells or both B cells andT cells, or are elevated relative to the level of B cells, T cells orboth B cells and T cells, in one or more control samples that caninclude, but are not limited to, samples from other cancer patients orfrom healthy, non-cancer patients. B cell, T cell, or B cell and T celllevels can be measured by a healthcare provider or by a clinicallaboratory, where a sample, e.g., a blood sample, is obtained from thepatient either by the healthcare provider or by the clinical laboratory.In one aspect, the patient's level of B cells, T cells or both B cellsand T cells, can be measured in a cytometric-based immunophenotypicassay.

In certain embodiments, this disclosure also provides a method oftreating a subject, e.g., a cancer patient, comprising administering tothe subject an effective amount of an isolated binding molecule thatspecifically binds to semaphorin-4D (SEMA4D) if Her2 and either PlexinB1 or Plexin B2 expression in a sample taken from the subject's tumorcells is above predetermined threshold levels, or is elevated relativeto the Her2 and either Plexin B1 or Plexin B2 expression in one or morecontrol samples. Her2, Plexin B1, and/or Plexin B2 expression in thesubject's tumor cells can be measured by a healthcare provider or by aclinical laboratory at the protein level and/or at the mRNA level. Incertain aspects, Her2, Plexin B1, and/or Plexin B2 expression can bemeasured in situ, e.g., via imaging techniques. In certain aspects Her2,Plexin B1, and/or Plexin B2 expression can be measured in a tumor cellsample obtained from the subject via a biopsy. In one aspect, Her2,Plexin B1, and/or Plexin B2 expression in tumor cellscan be measured inan immunoassay employing antibodies or antigen binding fragments thereofwhich recognize Her2, Plexin B1, and/or Plexin B2 proteins, orantigen-binding fragments, variants or derivatives thereof. In anotheraspect Her2, Plexin B1, and/or Plexin B2 expression can be measured viaa quantitative gene expression assay, e.g., an RT-PCR assay.

This disclosure also provides methods, assays, and kits to facilitate adetermination by a healthcare provider, a healthcare benefits provider,or a clinical laboratory to as to whether a subject, e.g., a cancerpatient, will benefit from treatment with either: (1) an effectiveamount of an isolated binding molecule that specifically binds tosemaphorin-4D (SEMA4D) and an effective amount of at least one otherimmune modulating therapy; or (2) an effective amount of an isolatedbinding molecule that specifically binds to semaphorin-4D (SEMA4D),where the subject has, or is suspected to have, tumor cells that areHer2+ and either Plexin B1+ or Plexin B2+. The methods, assays, and kitsprovided herein will also facilitate a determination by a healthcareprovider, a healthcare benefits provider, or a clinical laboratory to asto whether a subject, e.g., a cancer patient, will benefit fromtreatment with (1) an effective amount of an isolated binding moleculethat specifically binds to semaphorin-4D (SEMA4D) and an effectiveamount of at least one other immune modulating therapy; or (2) aneffective amount of an isolated binding molecule that specifically bindsto semaphorin-4D (SEMA4D) (e.g., where the subject's tumor cellsexpress, or can be determined to express, Her2 and either Plexin B1 orPlexin B2).

The present disclosure provides a method of treating a subject, e.g., acancer patient, comprising administering an effective amount of anisolated binding molecule that specifically binds to semaphorin-4D(SEMA4D) and an effective amount of at least one other immune modulatingtherapy; if the level of B-cells, T-cells, or T-cells and B-cells in asample taken from the patient is above a predetermined threshold level,or is above the level of B-cells, T-cells, or T-cells and B-cells in oneor more control samples. In some aspects, the sample is obtained fromthe patient and is submitted for measurement of the level of B-cells,T-cells, or T-cells and B-cells in the sample, for example, to aclinical laboratory.

Also provided is a method of treating a subject, e.g., a cancer patient,s comprising (a) submitting a sample taken from the subject formeasurement of the level of B-cells, T-cells, or T-cells and B-cells inthe sample; and, (b) administering an effective amount of an isolatedbinding molecule that specifically binds to semaphorin-4D (SEMA4D) andan effective amount of at least one other immune modulating therapy tothe subject if the subject's level of B-cells, T-cells, or T-cells andB-cells is above a predetermined threshold level, or is above the levelof B-cells, T-cells, or T-cells and B-cells in one or more controlsamples.

The disclosure also provides a method of treating a subject, e.g., acancer patient, comprising (a) measuring the level of B-cells, T-cells,or T-cells and B-cells in a sample obtained from a subject, e.g., acancer patient, wherein the subject's level of B-cells, T-cells, orT-cells and B-cells in the sample is measured, e.g., in acytometric-based immunophenotypic assay; (b) determining whether thelevel of B-cells, T-cells, or T-cells and B-cells in the sample is abovea predetermined threshold level, or is above the level of B-cells,T-cells, or T-cells and B-cells in one or more control samples; and, (c)advising, instructing, or authorizing a healthcare provider toadminister an effective amount of an isolated binding molecule thatspecifically binds to semaphorin-4D (SEMA4D) and an effective amount ofat least one other immune modulating therapy to the subject if thesubject's level of B-cells, T-cells, or T-cells and B-cells is above apredetermined threshold level, or is above the level of B-cells,T-cells, or T-cells and B-cells in one or more control samples.

In some aspects, the subject's level of B-cells, T-cells, or T-cells andB-cells can be measured in a cytometric-based immunophenotypic assay. Incertain aspects, the assay can be performed on a sample obtained fromthe subject, by the healthcare professional treating the patient, e.g.,using an assay as described herein, formulated as a “point of care”diagnostic kit. In some aspects, a sample can be obtained from thesubject and can be submitted, e.g., to a clinical laboratory, formeasurement of the level of B-cells, T-cells, or T-cells and B-cells inthe sample according to the healthcare professional's instructions,including but not limited to, using a cytometric-based immunophenotypicassay as described herein. In certain aspects, the clinical laboratoryperforming the assay can advise the healthcare provider or a healthcarebenefits provider as to whether the subject can benefit from treatmentwith an effective amount of an isolated binding molecule thatspecifically binds to semaphorin-4D (SEMA4D) and an effective amount ofat least one other immune modulating therapy, if the subject's level ofB-cells, T-cells, or T-cells and B-cells is above a predeterminedthreshold level, or is above the level of B-cells, T-cells, or T-cellsand B-cells in one or more control samples.

In certain aspects, results of an immunoassay as provided herein can besubmitted to a healthcare benefits provider for determination of whetherthe patient's insurance will cover treatment with an isolated bindingmolecule which specifically binds to semaphorin-4D (SEMA4D) and at leastone other immune modulating therapy.

Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering anti-SEMA4D binding molecules,e.g., antibodies, or antigen-binding fragments, variants, or derivativesthereof as a single agent or in combination with at least one otherimmune modulating therapy to a subject in need thereof are well known toor are readily determined by those skilled in the art. The route ofadministration of the anti-SEMA4D binding molecule, e.g., antibody, orantigen-binding fragment, variant, or derivative thereof as a singleagent or in combination with at least one other immune modulatingtherapy, can be, for example, oral, parenteral, by inhalation or topicalat the same or different times for each therapeutic agent. The termparenteral as used herein includes, e.g., intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, rectal, or vaginaladministration. While all these forms of administration are clearlycontemplated as being within the scope of the disclosure, an example ofa form for administration would be a solution for injection, inparticular for intravenous or intraarterial injection or drip. Asuitable pharmaceutical composition for injection can comprise a buffer(e.g. acetate, phosphate or citrate buffer), a surfactant (e.g.polysorbate), optionally a stabilizer agent (e.g. human albumin), etc.However, in other methods compatible with the teachings herein,anti-SEMA4D binding molecules, e.g., antibodies, or antigen-bindingfragments, variants, or derivatives thereof as a single agent or incombination with at least one other immune modulating therapy can bedelivered directly to the site of the adverse cellular populationthereby increasing the exposure of the diseased tissue to thetherapeutic agent.

As discussed herein, anti-SEMA4D binding molecules, e.g., antibodies, orantigen-binding fragments, variants, or derivatives thereof as a singleagent or in combination with at least one other immune modulatingtherapy can be administered in a pharmaceutically effective amount forthe in vivo treatment of diseases such as neoplastic disorders,including solid tumors. In this regard, it will be appreciated that thedisclosed binding molecules can be formulated so as to facilitateadministration and promote stability of the active agent. In certainembodiments, pharmaceutical compositions in accordance with the presentdisclosure comprise a pharmaceutically acceptable, non-toxic, sterilecarrier such as physiological saline, non-toxic buffers, preservativesand the like. For the purposes of the instant application, apharmaceutically effective amount of an anti-SEMA4D binding molecules,e.g., an antibody, or antigen-binding fragment, variant, or derivativethereof, as a single agent or in combination with at least one otherimmune modulating therapy shall be held to mean an amount sufficient toachieve effective binding to a target and to achieve a benefit, i.e., toinhibit, delay, or reduce metastases in a cancer patient.

The pharmaceutical compositions used in this disclosure comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include, e.g., water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Pharmaceutically acceptable carriers can include, but are notlimited to, 0.01-0.1 M, or 0.05 M phosphate buffer or 0.8% saline. Othercommon parenteral vehicles include sodium phosphate solutions, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives can also be present such as,for example, antimicrobials, antioxidants, chelating agents, and inertgases and the like.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition can be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and can be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of a certain particle size in the case ofdispersion and by the use of surfactants. Suitable formulations for usein the therapeutic methods disclosed herein are described in Remington'sPharmaceutical Sciences (Mack Publishing Co.) 16th ed. (1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. Incertain embodiments, isotonic agents, for example, sugars, polyalcohols,such as mannitol, sorbitol, or sodium chloride can be included in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., an anti-SEMA4D antibody, orantigen-binding fragment, variant, or derivative thereof, by itself orin combination with at least one other immune modulating therapy) in acertain amount in an appropriate solvent with one or a combination ofingredients enumerated herein, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle, which contains a basic dispersion medium and theother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, methods ofpreparation can include vacuum drying or freeze-drying, which can yielda powder of an active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof. The preparationsfor injections are processed, filled into containers such as ampoules,bags, bottles, syringes or vials, and sealed under aseptic conditionsaccording to methods known in the art. Further, the preparations can bepackaged and sold in the form of a kit. Such articles of manufacture canhave labels or package inserts indicating that the associatedcompositions are useful for treating a subject suffering from, orpredisposed to a disease or disorder.

Parenteral formulations can be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionscan be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

Certain pharmaceutical compositions can be orally administered in anacceptable dosage form including, e.g., capsules, tablets, aqueoussuspensions or solutions. Certain pharmaceutical compositions also canbe administered by nasal aerosol or inhalation. Such compositions can beprepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,and/or other conventional solubilizing or dispersing agents.

The amount of an anti-SEMA4D binding molecule, e.g., antibody, orfragment, variant, or derivative thereof, as a single agent or incombination with at least one other immune modulating therapy to becombined with the carrier materials to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. The composition can be administered as a single dose,multiple doses or over an established period of time in an infusion.Dosage regimens also can be adjusted to provide the optimum desiredresponse (e.g., a therapeutic or prophylactic response).

In keeping with the scope of the present disclosure, anti-SEMA4Dantibodies, or antigen-binding fragments, variants, or derivativesthereof as a single agent or in combination with at least one otherimmune modulating therapy can be administered to a human or other animalin accordance with the aforementioned methods of treatment in an amountsufficient to produce a therapeutic effect. The anti-SEMA4D antibodies,or antigen-binding fragments, variants or derivatives thereof as asingle agent or in combination with at least one other immune modulatingtherapy can be administered to such human or other animal in aconventional dosage form prepared by combining the antibody providedherein with a conventional pharmaceutically acceptable carrier ordiluent according to known techniques. It will be recognized by one ofskill in the art that the form and character of the pharmaceuticallyacceptable carrier or diluent is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. Those skilled in the art will furtherappreciate that a cocktail comprising one or more species of anti-SEMA4Dbinding molecules, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof as provided herein can be used.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of anti-SEMA4D binding molecule, e.g., antibody orantigen binding fragment, variant, or derivative thereof, as a singleagent or in combination with at least one other immune modulatingtherapy that when administered brings about a positive therapeuticresponse with respect to treatment of a patient with a disease to betreated, e.g., an inhibition, delay, or reduction of metastases in thepatient.

Therapeutically effective doses of the compositions of the presentdisclosure, for the inhibition, delay, or reduction of metastases, varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. In certain embodimentsthe patient is a human, but non-human mammals including transgenicmammals can also be treated. Treatment dosages can be titrated usingroutine methods known to those of skill in the art to optimize safetyand efficacy.

The amount of anti-SEMA4D binding molecule, e.g., antibody or bindingfragment, variant, or derivative thereof, administered as a single agentor in combination with at least one other immune modulating therapy isreadily determined by one of ordinary skill in the art without undueexperimentation given the disclosure of the present disclosure. Factorsinfluencing the mode of administration and the respective amount ofanti-SEMA4D binding molecule, e.g., antibody, antigen-binding fragment,variant or derivative thereof to be administered as a single agent or incombination with at least one other immune modulating therapy include,but are not limited to, the severity of the disease, the history of thedisease, the potential for metastases, and the age, height, weight,health, and physical condition of the individual undergoing therapy.Similarly, the amount of anti-SEMA4D binding molecule, e.g., antibody,or fragment, variant, or derivative thereof, as a single agent or incombination with at least one other immune modulating therapy to beadministered will be dependent upon the mode of administration andwhether the subject will undergo a single dose or multiple doses of thisagent.

The disclosure also provides for the use of an anti-SEMA4D bindingmolecule, e.g., antibody, or antigen-binding fragment, variant, orderivative thereof, as a single agent or in combination with at leastone other immune modulating therapy in the manufacture of a medicamentfor treating a subject with a cancer, wherein the medicament is used ina subject that has been pretreated with at least one other therapy. By“pretreated” or “pretreatment” is intended the subject has received oneor more other therapies (e.g., been treated with at least one othercancer therapy) prior to receiving the medicament comprising theanti-SEMA4D binding molecule, e.g., antibody or antigen-bindingfragment, variant, or derivative thereof as a single agent or incombination with at least one other immune modulating therapy.“Pretreated” or “pretreatment” includes subjects that have been treatedwith at least one other therapy within 2 years, within 18 months, within1 year, within 6 months, within 2 months, within 6 weeks, within 1month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week,within 6 days, within 5 days, within 4 days, within 3 days, within 2days, or even within 1 day prior to initiation of treatment with themedicament comprising the anti-SEMA4D binding molecule, for example, themonoclonal antibody VX15/2503 disclosed herein, or antigen-bindingfragment, variant, or derivative thereof as a single agent or incombination with at least one other immune modulating therapy. It is notnecessary that the subject was a responder to pretreatment with theprior therapy or therapies. Thus, the subject that receives themedicament comprising the anti-SEMA4D binding molecule, e.g., anantibody or antigen-binding fragment, variant, or derivative thereof asa single agent or in combination with at least one other immunemodulating therapy could have responded, or could have failed to respond(e.g., the cancer was refractory), to pretreatment with the priortherapy, or to one or more of the prior therapies where pretreatmentcomprised multiple therapies. Examples of other cancer therapies forwhich a subject can have received pretreatment prior to receiving themedicament comprising the anti-SEMA4D binding molecule, e.g., antibodyor antigen-binding fragment, variant, or derivative thereof as a singleagent or in combination with at least one other immune modulatingtherapy include, but are not limited to, surgery; radiation therapy;chemotherapy, optionally in combination with autologous bone marrowtransplant, where suitable chemotherapeutic agents include, but are notlimited to, those listed herein above; other anti-cancer monoclonalantibody therapy; small molecule-based cancer therapy, including, butnot limited to, the small molecules listed herein above;vaccine/immunotherapy-based cancer therapies; steroid therapy; othercancer therapy; or any combination thereof.

The practice of the present disclosure will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described are generally followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W. H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevier, Amsterdam); Goldsby et al., eds. (2000) KubyImmunology (4th ed.; H. Freeman & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlag); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall 2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Testing the Ability of an Anti-SEMA4D Antibody toDelay Tumor Growth in Immune Competent Mice

Experimental Design. The basic experimental design is as follows.Colon26 tumor cells were implanted subcutaneously into the flank ofsyngeneic immunocompetent Balb/c mice (5×10̂5 cells) or immune deficientSCID mice (1×10̂5 cells) in 0.2 ml saline. Treatment with Control Ig 2B8or anti-SEMA4D Ab 67 was initiated on day 2 post tumor implant. Mice(n=20) were treated twice weekly with 1.0 mg (approximately 50 mg/kg)each of monoclonal antibody via intraperitoneal (IP) injection. Tumorswere measured with calipers 3×/week starting 3 days post implant. Micewere weighed 2×/wk starting on day 3. Animals were sacrificed when tumorvolume reached 1000 mm3.

Anti-SEMA4D treatment delayed tumor growth in mice with competent immunesystem. Tumor growth was measured by calipers and measurements were usedto calculate tumor volume using the formula (w²×l)/2, where w=width,smaller measurement and l=length, in mm, of the tumor. Mean tumor volume(FIG. 1A) and Kaplan Meier survival curves (FIG. 1B), defined as time toendpoint where tumor volume=1000 mm3, are shown in FIGS. 1A and 1B.Statistical analysis was conducted using Two-way Analysis of Variance(ANOVA) and Log Rank analysis, respectively, which showed astatistically significant treatment effect with anti-SEMA4D antibody inBalb/c mice.

Twenty-nine percent (29%) tumor growth delay was achieved in Balb/cmice, however, no treatment related tumor growth delay was observed inSCID mice. Tumor growth delay (TGD), is defined as the increase in themedian time-to-endpoint (TTE) in a treatment group compared to thecontrol group: % TGD−[(T-C)/C]×100, where T=median TTE for a treatmentgroup, C=median TTE for the control group. The Balb/c animals treatedwith anti-SEMA4D antibody 67 showed a statistically significantreduction in primary tumor volume at the time of sacrifice over thecontrol animals (P<0.0001). This finding shows that the anti-SEMA4Dantibody was effective at delaying tumor growth in mice with a competentimmune system, but not in immune deficient mice.

Example 2 Testing the Ability of an Anti-SEMA4D Antibody to Delay TumorGrowth in Presence of CD8+ Effector T Cells

Experimental Design. Colon26 tumor cells were implanted subcutaneouslyinto the flank of Balb/c mice (5×10̂5 cells in 0.2 ml saline). Anti-CD8depleting antibody (Clone 2.43, BioXCell) or control Rat Ig (CloneLTF-2, BioXCell) (150 mg/kg) were administered via intraperitoneal (IP)injection on days −1, 0, 1, 11 and weekly thereafter. Treatment withControl Ig 2B8 or anti-SEMA4D Ab 67 was initiated on day 2. Mice (n=20)were treated twice weekly with 1.0 mg (approximately 50 mg/kg) ofmonoclonal antibody via intraperitoneal injection. Tumors were measuredwith calipers 3×/week starting 3 days post implant. Animals weresacrificed when the mean tumor volume of the control group reached 1000mm3, day 30 for Rat Ig treated groups, and day 26 for anti-CD8 treatedgroups.

Anti-SEMA4D treatment delayed tumor growth in presence of CD8+ Tlymphocytes. Tumor volume was measured by calipers using the formula(w²×l)/2, where w=width, smaller measurement and l=length, in mm, of thetumor. Statistical differences in tumor volume were determined using atwo-tailed One-Way Analysis of Variance (ANOVA) comparing antibodytreated groups with the Control Ig 2B8 group. Mean tumor volume areshown in FIG. 2.

Inhibition of tumor growth was also determined. Tumor growth inhibition(TGI) was measured using the following formula:%TGI=1−[(Tf−Ti)/mean(Cf−Ci)]; %TGI reported is the mean of %TGI for eachtreated tumor. Statistical differences in tumor volume were determinedusing a two-tailed One-Way Analysis of Variance (ANOVA) followed by theDunnett's multiple comparisons test comparing treated groups withcontrol 2B8 group. Thirty percent (30%) tumor growth inhibition wasachieved following treatment with anti-SEMA4D antibody, however notreatment related effect was observed when CD8+ T cells were depleted.These results show that tumor growth inhibition with anti-SEMA4D wasdependent on the presence of CD8+ effector T cells.

Example 3 Testing the Ability of an Anti-SEMA4D Antibody to IncreaseDensity of Tumor Infiltrating Lymphocytes (TIL)

Experimental Design. Colon26 tumor cells were implanted subcutaneouslyinto a flank of Balb/c mice (5×10̂5 cells in 0.2 ml saline). Treatmentwith Control Ig 2B8 or anti-SEMA4D Ab 67 was initiated on day 2 (50mg/kg IP, twice weekly, n=10). Tumors were measured with calipers3×/week starting 3 days post implant. Animals were sacrificed on day 27,when the mean tumor volume of the control group reached 1000 mm3.Tumors, including surrounding stroma and skin, were collected and fixedin formalin for 24 hours, then transferred to 70% ethanol. Samples werethen processed for paraffin embedding, and 5 micron sections were cutfrom the resulting blocks.

Adjacent sections were stained for Sema4D, CD8, and CD20 using thefollowing methods:

For Sema4D detection, slides were baked at 60° C. for 1 hour, thendeparaffinized and rehydrated through xylene and graded ethanol baths.Epitope retrieval was carried out by boiling 20-min with TargetRetrieval Solution (Dako, Carpinteria, Calif.) followed by 30-mincooling. Slides were washed twice with PBS containing 0.05% Tween-20(TPBS), then endogenous peroxidases were inactivated with a 10-min blockwith Dual Enzyme Block (Dako, Carpinteria, Calif.). Slides were washedwith TPBS twice, then nonspecific binding was blocked by a 20-minincubation with 2.5% normal goat serum in TPBS. Following a single TPBSwash, slides were incubated for 60 min with rabbit anti-Sema4D at 2μg/ml in TPBS, followed by 2 TPBS washes. Slides were then incubated for20 min with Envision HRP labeled goat anti-rabbit polymer (Dako,Carpinteria, Calif.) followed by 2 washes with TPBS and a 5-min DAB+incubation (Dako, Carpinteria, Calif.). Sections were counterstainedwith Harris hematoxylin, destained, blued with tap water, dehydrated,and non-aqueous mounted with Permount.

CD8 was detected using the method above, but using a commercial rabbitpolyclonal antibody (Abbiotec) at 2 μg/ml.

CD20 was detected using the method above, but using normal donkey serumfor blocking, and using a goat anti-CD20 primary antibody (Santa Cruz)at 1 μg/ml followed by a 20 minute incubation with HRP-labeled anti-goatantibody (Golden Bridge).

Slides were imaged at 20× magnification using a Retiga QICAM-12 bitcamera coupled to an Olympus Ix50 microscope.

Anti-SEMA4D treatment increased frequency of tumor infiltrating immunecells (TIL). Immune cell density was measured by scanning sections ofthe entire tumor, quantifying areas of CD8+ or CD20+ Tumor InfiltratingLymphocytes (TIL), and then normalizing to total tumor area. Sectionsfrom 9 (Control Ig) or 10 (anti-SEMA4D Ab 67) mice per group were usedfor analysis. Statistical significance was calculated for CD8 and CD20using two tailed unpaired T test to 95% CI.

Treatment of Colon26 tumors with anti-SEMA4D antibody 67 resulted in anincrease in both CD8+ T cell density and CD20+ T cell density, ascompared to the control group. The increase in density of the CD20+ Tcells was statistically significant to 95% with a P value of 0.0388. Theincrease in density of the CD8+ T cells showed a trend but was notstatistically significant. These findings show that anti-SEMA4Dtreatment of Colon26 tumors resulted in increased frequency of tumorinfiltrating immune cells. The results are shown graphically in FIGS. 3Aand 3B.

Example 4 Testing the Ability of an Anti-SEMA4D Antibody to AffectMigration and distribution of M1 and M2 Macrophage Subsets and CD8+ TCells at Leading Edge of Tumor

Anti-SEMA4D treatment altered macrophage and CD8+ T cell distribution atleading edge of tumor. Macrophage distribution was measured by scanningsections of the entire tumor, quantitating the area of M1 (staining withAlexa647 conjugated rat anti-F4/80 (Biolegend, clone BM8) at 2 μg/ml)and M2 (staining with biotin conjugated rat anti-CD206 (Biolegend, cloneC068C2) at 2 μg/ml), and then normalizing to total tumor area todetermine M1 and M2 density within the tumor. Sections from 9 (ControlIg) or 10 (anti-SEMA4D Ab 67) treated mice per group were used foranalysis. For determining cell density in the tumor growing front, a 300pixel width region (250 micron) was defined from the edge of the tumor.Statistical significance for M1 and M2 were calculated using one wayANOVA with Kruskal-Wallace and Dunn's post-hoc test to 95% CI. Change indensity of M1 macrophage normalized to leading edge of tumor wassignificant.

CD8+ T cell numbers were measured in whole tumor sections stained withanti CD8 antibody (Abbiotec Cat#250596 at 1:250) and DAB detectionsystem. The number of CD8+ events in entire tumor sections wereenumerated after thresholding for positive signal using ImageproSoftware. CD8+ density for each animal was calculated by dividing thenumber of CD8+ events by the whole tumor pixel area. Individual CD8densities were averaged to arrive at CD8+ T cell distribution in 2B8 andMAb 67 treated animals (n=10). Statistical significance was calculatedusing one way ANOVA with Kruskal-Wallace and Dunn's post hoc test to 95%CI.

SEMA4D distribution was measured by scanning sections of the entiretumor stained for SEMA4D with an antibody to an epitope distinct fromthat recognized by Ab 67 and analyzing for Sema4D distribution. Sectionsfrom 9 (Control Ig) or 10 (anti-SEMA4D Ab 67) treated mice per groupwere used for analysis.

Colon26 tumor cells expressed low levels of SEMA4D when cultured invitro, but upregulated SEMA4D in vivo at the leading edge of the tumor.This lead to establishment of a gradient of SEMA4D expression with highconcentration at the periphery of the tumor. Treatment with anti-SEMA4Dantibody neutralized SEMA4D and disrupted the gradient of expression.This resulted in a striking change in the migration and distribution ofmacrophage, as shown in FIG. 4A. In particular, tumors treated withanti-SEMA4D Ab 67 had higher levels of M1+ pro-inflammatory macrophagesat the leading edge to the tumor as shown in FIG. 4B. The increase inM1+ macrophage was statistically significant. Tumors treated withanti-SEMA4D Ab 67 also showed a decrease in the frequency of pro-tumorM2 macrophage at the leading edge of the tumor as shown in FIG. 4C.These findings showed that treatment with anti-SEMA4D Ab 67 alteredmacrophage distribution in a way that increased the density of tumorinhibitory macrophage, i.e., M1, at the leading edge of the tumor whiledecreasing the presence of tumor promoting macrophage, i.e., M2, in thatsame region. Furthermore, these findings showed an overall increase inthe CD8+ T cell density within tumors isolated from MAb 67-treated mice,as shown in FIG. 4D. These findings suggest that neutralization ofSEMA4D with MAb 67-2 facilitates entry of anti-tumor M1 macrophage intothe zone of highly proliferating tumor cells and CD8+ T cells throughoutthe zone and extending into the leading edge (inset).

Example 5 Testing the Ability of an Anti-SEMA4D Antibody to Delay TumorGrowth in Mice when used in Combination with Anti-CTLA4 Antibodies

Experimental Design. 5x10⁵ Colon26 tumor cells were implantedsubcutaneously into the flank of female Balb/c mice. Treatment withcontrol Mouse IgG1/2B8 or anti-SEMA4D/MAb 67-2 was initiated 1 day postinoculation (50 mg/kg, IP, weekly ×5), with or without anti-CTLA4/MAbUC10-4F10-11 (100 μg on day 8 and 50 μg on days 11 and 14 post tumorinoculation). Treatment with anti-PD1/RMP1-14 was initiated 1 day postinoculation (100 μg on day 3, twice weekly) in combination withanti-CTLA4/MAb UC10-4F10-11. There were 20 mice per group. Tumors weremeasured with calipers 2×/week starting 5 days post implant. Animalswere sacrificed when tumor volume reached 1000 mm3.

Combination of anti-SEMA4D and anti-CTLA4 antibodies delayed tumorgrowth in mice. Tumor growth was measured by calipers and measurementswere used to calculate tumor volume using the formula (w²×l)/2, wherew=width, smaller measurement and l=length, in mm, of the tumor. Meantumor volume and Kaplan Meier survival curves, defined as time toendpoint where tumor volume=1000 mm3, are shown in FIGS. 5A and 5B,respectively. Statistical analysis was conducted using Two-way Analysisof Variance (ANOVA) and Log Rank analysis, respectively, which showed astatistically significant treatment effect with anti-SEMA4D antibody (9%Tumor Growth Delay, TGD**) and anti-CTLA4 antibody (2% TGD, ns), and ahighly significant increase in tumor growth delay with the combinationof anti-SEMA4D and anti-CTLA4 antibodies (maximal TGD, 114%****). Theresponses were durable for at least 60 days.

The frequency of tumor regressions in Colon 26 tumor model was alsodetermined. Regression is the lack of palpable tumor, defined by a tumormeasuring <50 mm3 for at least two consecutive measurements. As shown inFIG. 5C, combination of anti-SEMA4D and anti-CTLA4 antibodies increasesthe number of regressions in Colon26 tumor model. Regressions for thecombination therapy (anti-SEMA4D+ anti-CTLA4 antibodies) arestatistically significant compared to Control Ig (p<0.0001) and comparedto anti-CTLA4 or anti-SEMA4D monotherapies (p=0.0022), as determined byFisher's Exact test. Importantly, these finding show that thecombination of anti-SEMA4D and anti-CTLA4 antibodies was synergistic:that is the combination was significantly more effective, resulting inincreased frequency of durable tumor regressions, than treatment withanti-SEMA4D antibody alone or with anti-CTLA4 antibody alone.Furthermore, these results demonstrate that the combination ofanti-SEMA4D and anti-CTLA4 antibodies is at least as effective as, orbetter than, the combination of anti-PD1 and anti-CTLA4.

It was further determined that treatment with anti-SEMA4D antibodyincreases tumor-reactive CTL activity, and also enhancesanti-CTLA4-mediated CTL activity. A follow-up study was conducted toexamine the effect of anti-CTLA monotherapy, compared to anti-CTLA4 andanti-SEMA4D combination therapy on the frequency of tumor-specific tumorinfiltrating leukocytes (TIL) and secretion of pro-inflammatorycytokines. In this follow-up study, immune cells were isolated fromtumors and spleens of Colon26 tumor-bearing mice treated in vivo withControl IgGl/Mab 2B8, anti-CTLA4/Mab UC10-4F10, or the combination ofanti-CTLA4/Mab UC10-4F10 and anti-SEMA4D/Mab 67. Tissues were harvestedon day 15, 1 day post final anti-CTLA4 antibody dose and just prior totumor regression. Total CD45+ TIL were assessed for secreted cytokinelevels, and frequency of IFNγ secreting CD8+ T cells in the presence ofMHC-I-restricted Colon26 tumor-specific immunodominant gp70 peptide wasdetermined by ELISPOT. The frequency of MEW I specific responders werecalculated by subtracting the media control from the peptide-containingwells.

As shown in FIG. 5D, increased levels of pro-inflammatory cytokines IFNγwas observed in the tumors of mice treated with anti-CTLA4 antibodymonotherapy (p=0.0135), which was further and significantly enhancedfollowing treatment with the combination therapy of anti-CTLA4 andanti-SEMA4D (p=0.0002 compared to control or monotherapy). In FIG. 5E,increased frequency of peptide-specific IFNγ secreting responders wasobserved in the spleens of mice treated with anti-CTLA4 antibody. Thisfinding was expected because anti-CTLA4 is reported to induce T cellactivation in the periphery. Combination therapy of anti-CTLA4 andanti-SEMA4D was not found to further enhance activity in the spleen. Incontrast, a substantial increase in frequency of peptide-specific IFNγsecreting responders was observed in the TIL following treatment withanti-CTLA4 monotherapy, which was further and significantly enhancedfollowing treatment with anti-CTLA4 and anti-SEMA4D combination therapy.This finding suggests that the addition of anti-SEMA4D treatment cansignificantly improve the tumor-specific CD8+ T cell activity in alocalized tumor-specific manner.

Example 6 Testing the Ability of an Anti-SEMA4D Antibody to Affect TumorInfiltration of Tumor-Specific Cytotoxic CD8+ T Cells

MAb 67-2 treatment increases the frequency of tumor-specific TIL andsecretion of pro-inflammatory cytokines. Following four weeks of in-vivoanti-SEMA4D treatment, tumors were dissociated and enriched for CD45+cells by magnetic separation. CD45+ TIL, pooled from 5 mice, wereincubated in the presence and absence of immunodominant tumor peptide,AH-1, at various cell densities. IFNγ secreting cells were measured byELISPOT; peptide specific response was determined by subtracting averageof wells without peptide. Each sample was tested in replicates of 6 andis graphed above. Statistical significance was determined withMann-Whitney non-parametric t test.

An increase in IFNγ secreting cells was observed in MAb 67-treated miceboth in the presence and absence of peptide, as shown in FIG. 6A. CD45+TIL, especially MHC-I-restricted peptide-specific CD8+ cytotoxic Tcells, represents activated effector cells following treatment with MAb67-2. FIG. 6B shows representative ELISPOT images. CD45+ TIL then werecultured ex vivo for 48 hr and assayed for cytokine secretion using CBAanalysis. As shown in FIG. 6C, Mab 67-2 promotes secretion of anti-tumorcytokines, such as IFNγ and TNFα, in TIL. Statistical significance wasdetermined with Mann-Whitney non-parametric t test.

A follow-up study was conducted to examine the effect of MAb 67-2treatment on the frequency of tumor-specific tumor infiltratingleukocytes (TIL) and secretion of pro-inflammatory cytokines. In thisfollow-up study, immune cells were isolated from tumors of Colon26tumor-bearing mice treated in vivo with Control IgG1/Mab 2B8 oranti-SEMA4D/Mab 67. Total CD45+ TIL were assessed for secreted cytokinelevels, and frequency of IFNγ secreting CD8+ T cells in the presence ofMHC-I-restricted Colon26 tumor-specific immunodominant gp70 peptide wasdetermined by ELISPOT. The frequency of MHC I specific responders werecalculated by subtracting the media control from the peptide-containingwells.

As shown in FIG. 6D, increased levels of pro-inflammatory cytokines IFNγand TNFα was observed in the TIL of mice treated with anti-SEMA4Dantibody. Furthermore, as shown in FIG. 6E, increased frequency ofpeptide-specific IFNγ secreting responders was observed in the TIL ofmice treated with anti-SEMA4D antibody. This finding suggests that theaddition of anti-SEMA4D treatment can significantly improve thetumor-specific CD8+ T cell activity in a localized tumor-specificmanner.

Example 7 Testing the Ability of an Anti-SEMA4D Antibody to Delay TumorGrowth in Mice when used in Combination with Anti-PD1 Antibodies

Experimental Design. 5×105 Colon26 tumor cells were implantedsubcutaneously into the flank of female Balb/c mice. Treatment withcontrol Mouse IgG1/2B8 or anti-SEMA4D/MAb 67-2 was initiated 1 day postinoculation (50 mg/kg, IP, weekly) Each group of mice was also treatedwith either control rat-Ig or rat anti-PD1/MAbRMP1-14 (100 μg, twice perweek, ×2 weeks starting at 3 days post tumor inoculation). There were 20mice per group. Tumors were measured with calipers 3×/week starting 5days post implant. Animals were sacrificed when tumor volume reached1000 mm3.

Combination of anti-SEMA4D and anti-PD1 antibodies delayed tumor growthin mice. Tumor growth was measured by calipers and measurements wereused to calculate tumor volume using the formula (w²×l)/2, wherew=width, smaller measurement, and l=length, in mm, of the tumor. Meantumor volume and Kaplan Meier survival curves, defined as time toendpoint where tumor volume=1000 mm3, are shown in FIGS. 7A and 7B,respectively. Statistical analysis was conducted using Two-way Analysisof Variance (ANOVA) and Log Rank analysis, respectively, which showed astatistically significant treatment effect with anti-SEMA4D antibodycombined with anti-PD1 antibody in Balb/c mice. These finding show thatthe combination of anti-SEMA4D and anti-PD1 antibodies was moreeffective than treatment with anti-SEMA4D or with anti-PD1 antibodyalone.

The frequency of regressions in Colon 26 tumor model was also measuredand is shown in FIGS. 7C and 7D. Regression is the lack of palpabletumor, defined by a tumor measuring <50 mm3 for at least two consecutivemeasurements. Combination of anti-SEMA4D and anti-PD1 antibodiesincreases the number of regressions in Colon26 tumor model. Regressionsfor the combination therapy (αSEMA4D+ α PD1 antibodies) arestatistically significant compared to Control Ig (p=0.0083) or singleagent anti-PD-1 (p=0.02), as determined by Fisher's Exact test.

Example 8 Testing the Ability of an Anti-SEMA4D Antibody to Delay TumorGrowth in Mice when used in Combination with Cyclophosphamide

Experimental Design. 5x105 Colon26 tumor cells were implantedsubcutaneously into the flank of female Balb/c mice. Treatment withcontrol Mouse IgG1/2B8 or anti-SEMA4D/MAb 67-2 was initiated 1 day postinoculation (50 mg/kg, IP, weekly). Treatment with cyclophosphamide (50mg/kg, IP) was administered on days 12 and 20. There were 20 mice pergroup. Tumors were measured with calipers 3×/week starting 5 days postimplant. Animals were sacrificed when tumor volume reached 1000 mm3.

Combination of anti-SEMA4D antibodies and cyclophosphamide delayed tumorgrowth in mice. Tumor growth was measured by calipers and measurementswere used to calculate tumor volume using the formula (w²×l)/2, wherew=width, smaller measurement, and l=length, in mm, of the tumor. Meantumor volume, median tumor volume, and Kaplan Meier survival curves,defined as time to endpoint where tumor volume=1000 mm3, are shown inFIGS. 8A, 8B and 8C, respectively. Statistical analysis was conductedusing Two-way Analysis of Variance (ANOVA) and Log Rank analysis,respectively, which showed a statistically significant treatment effectwith anti-SEMA4D antibody combined with cyclophosphamide in Balb/c mice.

Specifically, the findings show a 232% Tumor Growth Delay (TGD) whenanti-SEMA4D antibodies were used in combination with cyclophosphamide.This finding was statistically significant compared to Control Ig(p<0.0001), as determined by Mantel Cox Log Rank analysis. There wasalso a 3% TGD when anti-SEMA4D antibody treatment was used alone(statistically significant compared to Control Ig (p=0.0282)) and a 96%TGD when cyclophosphamide treatment was used alone (statisticallysignificant compared to Control Ig (p<0.0001), as determined by MantelCox Log Rank analysis. The responses were durable for at least 81 days.These finding show that the combination of anti-SEMA4D antibodies andcyclophosphamide was more effective at delaying tumor growth thantreatment with anti-SEMA4D antibody alone or cyclophosphamide alone.

The frequency of regression in Colon 26 tumor model was also measuredand shown in FIGS. 8D and 8E. Regression is the lack of palpable tumor,defined by a tumor measuring <50 mm3 for at least two consecutivemeasurements. Combination of anti-SEMA4D antibodies and cyclophosphamideincreases the number of regressions in Colon26 tumor model. Regressionsfor the combination therapy (αSEMA4D antibodies+cyclophosphamide) arestatistically significant compared to Control Ig (p<0.003), asdetermined by Fisher's Exact test. These data demonstrate increasedefficacy and response to treatment with cyclophosphamide when combinedwith anti-SEMA4D antibody.

Example 9 Testing the Ability of an Anti-SEMA4D Antibody to Delay TumorGrowth in Mice when used in Combination with Anti-HER2/neu Antibodies

Experimental Design. 3×10⁴ Tubo.A5 tumor cells were implantedsubcutaneously into the mammary fat pad of female Balb/c mice. Treatmentwith control Mouse IgG1/2B8.1E7 or anti-SEMA4D/MAb 67-2 was initiated 7days post inoculation (50 mg/kg, IP, weekly ×6). Treatment withanti-Neu/MAb7.16.4 (200 μg IP weekly ×2, initiated when tumor volume wasapproximately 200 mm3, on days 21 and 28). There were 15 mice per group.Tumors were measured with calipers 2×/week starting 11 days postimplant. Animals were sacrificed when tumor volume reached 800 mm3.

Combination of anti-SEMA4D and anti-HER2/Neu antibodies delayed tumorgrowth in mice. Tumor growth was measured by calipers and measurementswere used to calculate tumor volume using the formula (w²×l)/2, wherew=width, smaller measurement, and l=length, in mm, of the tumor. Meantumor volume and Kaplan Meier survival curves, defined as time toendpoint where tumor volume=800 mm3, are shown in FIGS. 9A and 9B,respectively. Statistical analysis was conducted using Two-way Analysisof Variance (ANOVA) and Log Rank analysis, respectively, which showed astatistically significant treatment effect with anti-SEMA4D antibodycombined with anti-Her2/Neu antibody in Balb/c mice. The findings show48% Tumor Growth Delay when anti-SEMA4D antibody is used in combinationwith anti-Neu antibody and that this is statistically significantcompared to using an irrelevant control antibody (p=0.017) or anti-Neumonotherapy (p=0.006), as determined by Mantel Cox Log Rank analysis.

The frequency of tumor regressions in Tubo tumor model was also measuredand is shown in FIG. 9C. Regression is the lack of palpable tumor,defined as a tumor measuring <50 mm3 for at least two consecutivemeasurements. Combination of anti-SEMA4D and anti-Neu antibodiesincreases the number of regressions in Tubo-bearing mice. Regressionsfor the combination therapy (αSEMA4D+αNeu antibodies) are statisticallysignificant compared to Control Ig (p =0.016), as determined by Fisher'sExact test.

Example 10 Testing the Ability of an Anti-SEMA4D Antibody to DelayGrowth of in vivo Mammary Carcinoma Model

Experimental Design. 3×104 Tubo.A5 tumor cells were implantedsubcutaneously into the mammary fat pad of female Balb/c mice. Treatmentwith control Mouse IgG1/2B8.1E7 or anti-SEMA4D/MAb 67-2 was initiated 6days post inoculation (50 mg/kg, IP, weekly ×6). There were 20 mice pergroup, however, some mice were excluded from analysis due to prematuredeath before reaching endpoint resulting from ulceration or general illhealth. Tumors were measured with calipers 2×/week starting 13 days postimplant. Animals were sacrificed when tumor volume reached 800 mm3.

Anti-SEMA4D antibody treatment delayed tumor growth in mice. Tumorgrowth was measured by calipers and measurements were used to calculatetumor volume using the formula (w²×l)/2, where w=width, smallermeasurement, and l=length, in mm, of the tumor. Mean tumor volume andKaplan Meier survival curves, defined as time to endpoint where tumorvolume=800 mm3, are shown in FIGS. 10A and 10B, respectively.Statistical analysis was conducted using Two-way Analysis of Variance(ANOVA) and Log Rank analysis, respectively, which showed astatistically significant treatment effect with anti-SEMA4D antibody.The findings show maximal Tumor Growth Delay (133%) with anti-SEMA4Dantibody treatment; this is statistically significant compared to usingan irrelevant control antibody (p<0.0001), as determined by Mantel CoxLog Rank analysis.

The frequency of tumor regressions in the Tubo.A5 tumor model was alsomeasured and is shown in FIG. 10C-10E. Regression is the lack ofpalpable tumor, defined as a tumor measuring <50 mm3 for at least twoconsecutive measurements. At 90 days post implant, 85% (12/14) of Mab67-treated mice were tumor-free regressors and one of the 14 had neverdeveloped measurable tumor, compared to 0/14 regressions in the micetreated with Control Ig. On day 90, mice who had completely rejectedtheir primary tumors (13/14 of Mab 67-treated mice) were challenged withviable Tubo.A5 (30,000) on contralateral side; naive mice were includedas controls for graft. As shown in FIG. 10D, all 13 mice that weretreated with anti-SEMA4D rejected subsequent tumor challenge, suggestingan immunologic memory response, in contrast to naive mice who did notreject the tumor challenge as shown in FIG. 10E. The regressionfrequency is statistically significant compared to Control Ig(p<0.0001), as determined by Fisher's Exact test.

Example 11 Effect of Anti-SEMA4D Antibody on T Cell Infiltration andMDSC in Tubo.A5 Tumor Models

Experimental Design. Tubo.A5 tumors were implanted into syngeneic BALB/cmice. Treatment with murine control Ig or anti-SEMA4D MAb 67 wasinitiated on day 6 (50 mg/kg IP, weekly). Tumors were harvested on day39, just prior to tumor regression. FACS was performed on Lymphocytecell fractions pooled from tumors of 14-21 mice/group. Mean of assayreplicates are shown; significance was determined using two-tailedt-test.

As shown in FIGS. 11A and 11B, anti-SEMA4D antibody therapy increasesCD3+ T cell infiltration and decreases CD11b+Gr1+ MDSC in tumors of micetreated with anti-SEMA4D. These data suggest an increase inanti-tumorigenic T cell response and a decrease in immunosuppressivecells, such as MDSC. These data are consistent with the modulation ofthe immune balance observed in the Colon26 model.

Example 12 Dose Titration of Mab 67 in Tubo.A5 and Colon26 Tumor Models

Experimental Design for Tubo.A5 Tumor Model. 3x104 Tubo.A5 tumor cellswere implanted subcutaneously into the mammary fat pad of female Balb/cmice. Treatment with control Mouse IgG1/2B8.1E7 (50 mg/kg, IP, weekly×6)or anti-SEMA4D/MAb 67-2 (1, 10 or 50 mg/kg, IP, weekly ×6) was initiated6 days post inoculation. There were at least 20 mice per group, however,some mice were excluded from analysis due to premature death beforereaching endpoint resulting from ulceration or general ill health.Tumors were measured with calipers 2×/week starting 13 days postimplant. Animals were sacrificed when tumor volume reached 800 mm3.

Experimental Design for Colon26 Tumor Model. 5×105 Colon26 tumor cellswere implanted subcutaneously into the flank of female Balb/c mice.Treatment with control Mouse IgG1/2B8.IE7 (50 mg/kg, IP, weekly×5) oranti-SEMA4D/MAb 67-2 (0.3, 3, 10, or 50 mg/kg, IP, weekly ×5) wasinitiated 1 day post inoculation, with or without anti-CTLA4/MAbUC10-4F10-11 (100 μg ˜5 mg/kg on day 8, and 50 μis ˜2.5 mg/kg on days 11and 14 post tumor inoculation). There were 15 mice per group. Tumorswere measured with calipers 2×/week starting 5 days post implant.Animals were sacrificed when tumor volume reached ≧1000 mm3.

Minimal Effective Dose is Approximately 3 mg/kg. Treatment of Tubo.A5tumor with 50 or 10 mg/kg Mab 67 resulted in tumor growth delay that wasstatistically significant compared to control IgG (p<0.0001 and p=0.0015respectively), but not significantly different from one another.Regression frequencies of 38% (9/24) and 54% (6/13) in Tubo.A5 tumortreated with 50 or 10 mg/kg Mab 67 were also significantly significant(p=0.0069 and p=0.0014). In contrast, 1 mg/kg Mab 67 was ineffective anddid not significantly delay tumor growth (p=0.01441). In this model, theminimal effective dose was determined to be between 1 and 10 mg/kg.Tumor growth was measured by calipers and measurements were used tocalculate tumor volume using the formula (w²×l)/2, where w=width,smaller measurement, and l=length, in mm, of the tumor. Mean tumorvolume and Kaplan Meier survival curves, defined as time to endpointwhere tumor volume=800 mm3, are shown in FIGS. 12A and 12B,respectively. Statistical analysis was conducted using Two-way Analysisof Variance (ANOVA) and Log Rank analysis, respectively.

Further refinement of effective Mab 67 dose was investigated in theColon26 model and was determined to be ≧3 mg/kg. Treatment of Colon26tumors with anti-CTLA4 + anti-SEMA4D resulted in a maximal tumor growthdelay (119%) compared to anti-CTLA4 monotherapy when doses ofanti-SEMA4D/MAb 67 were >3 mg/kg; at 10 mg/kg MAB 67, p=0.0101 and at 3mg/kg, p=0.0571, as compared to anti-CTLA4 monotherapy and determinedusing Mantel Cox Log Rank analysis. All doses between 3-50 mg/kg werenot significantly different than one another. In contrast, whenanti-CTLA4 was administered in combination with 0.3 mg/kg MAB 67, thedifference was statistically different than treatment with 10 mg/kg MAb67 (p=0.0325) but was not statistically significant compared totreatment with anti-CTLA4 monotherapy (p=0.4945). Tumor growth wasmeasured by calipers and measurements were used to calculate tumorvolume using the formula (w²×l)/2, where w=width, smaller measurement,and l=length, in mm, of the tumor. Mean tumor volume and Kaplan Meiersurvival curves, defined as time to endpoint where tumor volume=1000mm3, are shown in FIGS. 12C and 12D, respectively. Statistical analysiswas conducted using Two-way Analysis of Variance (ANOVA) and Log Rankanalysis, respectively.

Example 13 Effect of Anti-SEMA4D Antibody in Delaying Tumor Growth inColon26 and Tubo.A5 Tumor Models

Experimental Design. FIG. 13 is a summary of experiments conducted inthe above Examples showing tumor regressions and growth after tumorre-challenge in Colon26 and Tubo.A5 tumor models. The experiment designof the respective experiments is summarized in the above Examples.

Anti-SEMA4D antibody therapy results in complete and durable tumorregressions. As shown in FIG. 13, treatment with anti-SEMA4D antibodytherapy results in a statistically significant increase in tumorregression when compared to treatment with control Mouse IgG1 in boththe Colon26 and Tubo.A5 models, 7% (P≦0.001***) and 85% (P≦0.0001****),respectively. Moreover, treatment with anti-SEMA4D antibody therapy isnot significantly different than treatment with anti-PD1 alone (7% withanti-SEMA4D alone vs. 8% with anti-PD1 alone, n.s.), but issignificantly enhanced when used in combination with anti-PD1 therapy(28% for combination therapy vs. 7% for anti-SEMA4D or 8% for anti-PD1monotherapy, P≦0.0001****). Furthermore, treatment with anti-SEMA4Dantibody therapy in combination with anti-CTLA4 therapy results in astatistically significant increase in tumor regression when compared totreatment with anti-CTLA4 alone (74% for combination therapy vs. 20% foranti-CTLA4 monotherapy, P≦0.0001****). Additionally, treatment withanti-SEMA4D antibody therapy in combination with anti-CTLA4 therapyresults in a statistically significant increase in tumor regression whencompared to treatment with anti-SEMA4D in combination with anti-PD1 (74%for anti-SEMA4D/anti-CTLA4 combination therapy vs. 60% foranti-SEMA4D/anti-PD1 combination therapy, P≦0.001****). The greaterapparent synergy between anti-SEMA4D in combination with anti-CTLA4 ascompared to anti-SEMA4D in combination with anti-PD1 indicates that notall immune checkpoint blockade inhibitors are equivalent in this regardand that differences in mechanism can be associated with differentialtherapeutic benefit. Lastly, treatment with anti-SEMA4D in combinationwith cyclophosphamide results in a statistically significant increase intumor regression when compared to treatment with cyclophosphamide alone(40% for combination therapy vs. 10% for cyclophosphamide monotherapy,P≦0.01**).

Many modifications and other embodiments of the embodiments set forthherein will come to mind to one skilled in the art to which thisdisclosure pertains, having the benefit of the teachings presented inthe foregoing descriptions and the associated drawings. Therefore, it isto be understood that the disclosure is not to be limited to thespecific embodiments disclosed and that modifications and otherembodiments are intended to be included within the scope of the appendedclaims and list of embodiments disclosed herein. Although specific termsare employed herein, they are used in a generic and descriptive senseonly and not for purposes of limitation.

What is claimed is:
 1. A method of increasing Tumor InfiltratingLeukocyte (TIL) frequency in a solid tumor microenvironment (TME),comprising administering to a subject with a solid tumor an effectiveamount of an isolated antibody or antigen-binding fragment thereof thatspecifically binds to semaphorin-4D (SEMA4D), wherein the antibody orfragment thereof comprises a variable heavy chain (VH) comprising VHCDRs1-3 comprising SEQ ID NOS: 6, 7, and 8, respectively, and a variablelight chain (VL) comprising VLCDRs 1-3 comprising SEQ ID NOS: 14, 15,and 16, respectively, wherein administration of the antibody or fragmentthereof can increase the frequency of TIL in the TME.
 2. The method ofclaim 1, wherein administration of the antibody or fragment thereofincreases the frequency of CD8+ T lymphocytes, CD20+ B lymphocytes, M1+macrophages, or a combination thereof in the TME.
 3. The method of claim1, wherein administration of the antibody or fragment thereof decreasesthe frequency of M2+ macrophages in the TME.
 4. The method of claim 2,wherein administration of the antibody or fragment thereof increases thedensity of CD20+ B lymphocytes in the TME.
 5. The method of claim 2,wherein administration of the antibody or fragment thereof increases thedensity of CD8+ T lymphocytes in the TME.
 6. The method of claim 5,wherein the CD8+ T lymphocytes comprise tumor-specific CD8+ Tlymphocytes.
 7. The method of claim 6, wherein the tumor-specific CD8+ Tlymphocytes can secrete interferon gamma (IFN-y), tumor necrosisfactor-alpha (TNFα), or a combination thereof.
 8. The method of claim 1,further comprising recovering TIL from the TME after administering theantibody or fragment thereof to the subject.
 9. The method of claim 8,further comprising expanding the recovered TIL in vitro.
 10. The methodof claim 9, wherein the in vitro expanding comprises incubating therecovered TIL with allo-reactive feeder cells, interleukin-2 (IL-2),anti-CD3 antibody, or any combination thereof.
 11. The method of claim9, wherein the in vitro expanding enriches for cytotoxic CD8+ T cells.12. The method of claim 9, further comprising transducing the TIL with achimeric antigen receptor.
 13. The method of claim 12, wherein thechimeric antigen receptor is reactive to a tumor antigen of the solidtumor.
 14. The method of claim 9, further comprising transferring theexpanded TIL back into the subject.
 15. The method of claim 14, furthercomprising administering IL-2 to the subject.
 16. The method of claim 1,wherein the antibody or antigen-binding fragment thereof inhibits SEMA4Dinteraction with its receptor.
 17. The method of claim 16, wherein thereceptor is Plexin-B1.
 18. The method of claim 1, wherein the antibodyor antigen-binding fragment thereof inhibits SEMA4D-mediated Plexin-B1signal transduction.
 19. The method of claim 1, wherein the solid tumoris of a cancer is selected from the group consisting of carcinoma,lymphoma, blastoma, sarcoma, squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung, squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastrointestinal cancer, gastric cancer, pancreatic cancer,neuroendocrine cancer, glioblastoma, cervical cancer, ovarian cancer,liver cancer, bladder cancer, brain cancer, hepatoma, breast cancer,colon cancer, colorectal cancer, endometrial or uterine carcinoma,esophageal cancer, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, head and neckcancer, and a combination thereof.
 20. The method of claim 1, whereinthe VH and VL comprise, respectively, SEQ ID NO: 9 and SEQ ID NO: 17, orSEQ ID NO: 10 and SEQ ID NO:
 18. 21. A method of improvingtumor-specific CD8+ T cell activity in a solid tumor microenvironment(TME), comprising administering to a subject with a solid tumor aneffective amount of an isolated antibody or antigen-binding fragmentthereof that specifically binds to semaphorin-4D (SEMA4D), wherein theantibody or fragment thereof comprises a variable heavy chain (VH)comprising VHCDRs 1-3 comprising SEQ ID NOS: 6, 7, and 8, respectively,and a variable light chain (VL) comprising VLCDRs 1-3 comprising SEQ IDNOS: 14, 15, and 16, respectively, wherein administration of theantibody or fragment thereof improves the tumor-specific CD8+ T cellactivity in the TME.
 22. The method of claim 21, wherein thetumor-specific CD8+ T lymphocytes can secrete interferon gamma (IFN-γ),tumor necrosis factor-alpha (TNFα), or a combination thereof.
 23. Amethod for inhibiting, delaying, or reducing tumor growth in a subjectwith cancer, comprising administering to the subject an effective amountof an isolated antibody or antigen-binding fragment thereof thatspecifically binds to semaphorin-4D (SEMA4D), in combination with aneffective amount of at least one other immune modulating therapy,wherein the antibody or antigen, wherein the antibody or fragmentthereof comprises a variable heavy chain (VH) comprising VHCDRs 1-3comprising SEQ ID NOS: 6, 7, and 8, respectively, and a variable lightchain (VL) comprising VLCDRs 1-3 comprising SEQ ID NOS: 14, 15, and 16,respectively, and wherein the at least one other immune modulatingtherapy comprises a cancer vaccine, an immunostimulatory agent, adoptiveT cell or antibody therapy, a regulatory T cell (Treg) modulator, or acombination thereof.
 24. The method claim 23, wherein the at least oneother immune modulating therapy comprises a regulatory T cell (Treg)modulator.
 25. The method of claim 23, further comprising administrationof cyclophosphamide.